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DENTAL  DEPARTMENT, 

UNIVERSITY  OF  CALIFORNIA. 


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PHARMACEUTICAL 
BACTERIOLOGY 


SCHNEIDER 


PHARMACEUTICAL 
BACTERIOLOGY 


WITH  SPECIAL  REFERENCE  TO 

DISINFECTION  AND  STERILIZATION 


JIM 


BY 


ALBERT  SCHNEIDER,  M.  D.,  Ph.  D. 

.    =      (Columbia  University) 

PROFESSOR  OF   PHARMACOGNOSY,   HISTOLOGY,  AND   BACTERIOLOGY,   CALIFORNIA    COLLEGE  OF 

PHARMACY;  PHARMACOGNOSIST,  u.  s.  DEPARTMENT  OF  AGRICULTURE. 


WITH  86  ILLUSTRATIONS 


PHILADELPHIA 

P.    BLAKISTON'S   SON   &   CO. 

1012   WALNUT   STREET 
1912 


COPYRIGHT,  1912,  BY  P.  BLAKISTON'S  SON   &  Co. 


Printed   by 

The  Maple  Press 

York,  Pa. 


PREFACE. 


The  recent  growth  and  development  of  the  professional  side  of  pharmacy 
has  made  new  text-books  necessary.  The  present  volume  is  the  product  of 
such  progress. 

The  illustrations  have  been  selected  with  a  view  to  a  fuller  explanation 
of  the  text.  The  descriptions  of  the  illustrations  have  been  made  unusually 
complete.  This  is  to  make  it  possible  for  the  student  to  ascertain  the  use  of 
every  article  illustrated  without  the  necessity  of  searching  for  additional 
information  in  the  text  itself.  Some  of  the  illustrations  are  from  original 
drawings,  others  are  from  electros  supplied  by  the  Bausch  &  Lomb 
Optical  Company  and  the  Cutter  Biological  Laboratory  of  Berkeley, 
California.  Still  others  are  taken  from  recent  works  on  bacteriology, 
notably  Williams'  "Manual  of  Bacteriology." 

Attempts  have  been  made  to  adhere  strictly  to  the  subject  from  the 
standpoint  of  the  pharmacist,  with  only  enough  treatment  of  general 
bacteriology  to  make  clear  the  collateral  relationships,  especially  as  it 
pertains  to  medical  and  commercial  or  industrial  bacteriology. 

While  this  volume  is  primarily  intended  for  students  in  colleges  of 
pharmacy,  it  is  hoped  it  will  also  be  found  useful  by  practising  pharmacists. 

SAN  FRANCISCO. 


CONTENTS. 


PAGE. 

CHAPTER  I. — GENERAL  INTRODUCTION. i 

Introduction  of  bacteriology  into  colleges  of  pharmacy.  Relationship  of 
pharmaceutical  and  medical  bacteriology.  Reasons  why  pharmacists  should 
study  bacteriology. 

CHAPTER  II.— HISTORICAL 5 

Introduction.  From  Hippocrates  (300  B.  C.)  to  Leeuwenhoek  (1656),  the 
earliest  ideas  regarding  infections,  epidemics  and  spontaneous  generation.  From 
Leeuwenhoek  (1656)  to  Schwann  (1837),  the  discovery  of  micro-organisms  and 
the  earliest  observations  regarding  their  activities.  From  Schwann  (1837)  to 
Pasteur  (1862),  the  earlier  investigations  pertaining  to  the  relationship  of  micro- 
organisms to  fermentation  and  to  disease.  From  Pasteur  (1862)  to  Behring 
(1890),  period  of  remarkable  activity  in  bacteriological  pathology,  listerism, 
antiseptic  surgery,  etc.  From  Behring  (1890)  to  Wright  (1907),  discovery  of 
serum  therapy,  bacterial  vaccines  and  development  of  utilitarian  bacteriology. 

CHAPTER  III. — GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  OF  BACTERIA  ....     21 
Classification  of  microbes.     General  morphology.     General  physiology. 

CHAPTER  IV. — RANGE  AND  DISTRIBUTION  OF  BACTERIA 33 

Bacteria  of  earth,  air  and  water.  Bacteria  found  in  animals,  in  plants,  on  non- 
living objects,  etc.  Altitudinal  range.  Latitudinal  range. 

CHAPTER  V.— BACTERIOLOGICAL    TECHNIC 36 

Cleaning  glassware.  Plugging  containers  with  cotton.  Filling  test-tubes  with 
culture  media.  Preparation  of  culture  media.  Sterilization  of  culture  media. 
Neutralization  of  culture  media.  Making  bacterial  cultures.  Making  bacterial 
counts.  Preparing  bacterial  stains.  Examining  bacteria. 

CHAPTER  VI.— BACTERIA  IN  THE  INDUSTRIES 93 

The  function  of  bacteria  in  agriculture.  Bacteria  in  milk  and  in  the  dairying 
industry.  Bacterial  pest  exterminators.  Bacteria  in  the  tanning  industry. 
Rotting  bacteria.  Cider  making. 

CHAPTER  VII. — IMMUNITY,    BACTERIAL  ACTIVITIES  AND   BACTERIAL  PRODUCTS  114 
Immunity,  natural  and  acquired.     Race,  age  and  sex  immunity.     Anaphylaxis. 
Phagocytosis.     Ehrlich's  side-chain  theory.     Toxins  and  antitoxins.     Agglutin- 
ins.     Precipitins.     Lysins.     Opsonins  and  the  opsonic  index. 

CHAPTER  VIII. — THE  MANUFACTURE  AND  USE  OF  SERA  AND  VACCINES  .    .    .   125 
Antidiphtheric    serum.     Other  sera.     Bacterial  vaccines  (bacterins).      Concen- 
trated diphtheria  antitoxin.     Manufacture  of  bacterial  vaccines.     Tuberculins. 
Small-pox  vaccine.     Rabies  vaccine. 

vii 


Vlll  CONTENTS. 

PAG* 

CHAPTER  IX.— YEASTS  AND  MOULDS 142 

Yeast  organisms.  Moulds.  Organisms  active  in  fermented  drinks,  as  beer, 
wine  and  sake.  Yeast  cakes. 

CHAPTER  X.— PROTOZOA  IN  DISEASE ' 156 

Rhizopoda.     Flagellata.     Sporozoa. 

CHAPTER    XI. — DISINFECTANTS    AND     DISINFECTION.      FOOD    PRESERVATIVES. 

INSECTICIDES 159 

Principles  of  sterilization  and  disinfection.  Pasteurization.  Standardization  of 
disinfectants.  Methods  of  sterilization.  Disinfectants  and  their  use.  Disin- 
fection of  rooms  and  buildings.  Disinfection  of  sewage.  Sterilization  of  water 
supplies.  Food  preservatives,  their  use  and  abuse.  Insecticides  and  other  pest 
exterminators. 

CHAPTER  XII.— STERILIZATION  AND  DISINFECTION  IN  THE  PHARMACY      ....   190 
Sterilization  of  containers,  stoppers,  etc.     Surgical  supplies.     Dusting  powders. 
Salves  and  pastes.     Gargles,  washes,  etc.     Chemicals  and  galenicals.     Solutions 
for  hypodermic  use.     Ampuls. 

CHAPTER  XIII.— COMMUNICABLE  DISEASES  WITH  SUGGESTIONS  ON  PREVENTIVE 

MEDICINE 202 

Causes  of  disease.  Tuberculosis.  Typhoid  fever.  Pneumonia.  Small-pox. 
Malaria.  Diphtheria.  Cancer.  Plague.  Asiatic  cholera.  Yellow  fever.  Pel- 
lagra. Syphilis.  Gonorrhea.  Tabulation  of  diseases. 

CHAPTER  XIV. — A  BACTERIOLOGICAL  AND  MICROSCOPICAL  LABORATORY  FOR  THE 

PHARMACIST 225 

Position  of  laboratory.  Size  of  laboratory.  Furnishings.  Equipment.  Appa- 
ratus. Reference  library.  Outline  of  microscopical  and  bacteriological  work. 

GENERAL  INDEX 231 


PHARMACEUTICAL    BACTERIOLOGY. 


CHAPTER  I. 
GENERAL  INTRODUCTION. 

In  introducing  the  first  of  a  new  series  of  text-books,  certain  explanations 
are  necessary  or  at  least  desirable,  which,  after  the  subject  is  well  established, 
become  superfluous.  Comparatively  speaking,  the  science  of  bacteriology 
is  not  new,  but  its  introduction  into  pharmacy  is  of  very  recent  date. 

Medical  bacteriology  forms  the  very  framework  of  medical  practice.  It 
has  brought  about  our  modern  antiseptic  surgery  which  has  been  the  means 
of  saving  countless  lives.  It  has  led  to  the  still  more  recent  discoveries  in 
serum  therapy  and  the  opsonic  theory  of  disease. 

About  1896  a  few  of  the  colleges  of  pharmacy  in  the  United  States  gave 
optional  courses  of  instruction  in  bacteriology.  At  the  present  time  nearly 
all  of  the  leading  colleges  of  pharmacy  give  instruction  in  bacteriology  and 
in  many  of  these  institutions  the  courses  are  compulsory,  forming  a  part  of 
the  prescribed  curriculum,  represented  by  lectures  and  laboratory  work.  In 
some  universities  the  students  of  pharmacy  receive  their  bacteriological 
instruction  in  the  department  of  medicine  or  perhaps  dentistry.  However, 
pharmaceutical  bacteriology  and  medical  bacteriology  are  quite  distinct. 
Medical  students  study  this  subject  from  the  standpoint  of  pathology  and 
disease,  matters  which  concern  the  pharmacist  but  little.  Students  of 
pharmacy  do  not  have  the  time  necessary  to  devote  themselves  extensively 
to  special  laboratory  methods  and  technic,  nor  is  it  advisable  that  they 
should  receive  extensive  laboratory  instruction  in  pathology.  Pharmaceutical 
bacteriology  must  be  suitably  adapted  to  the  practice  of  pharmacy. 

The  pharmacist  should  have  a  fair  knowledge  of  general  bacteriology,  in 
order  that  he  may  realize  what  important  relationships  bacteria  bear  to 
human  activities  in  general,  to  medical  practice  more  especially,  and  in  order 
that  he  may  comprehend  quite  fully  the  significance  of  these  minute  organ- 
isms in  pharmaceutical  practice.  He  should  know  what  pharmaceutical  prep- 
arations and  what  medicinal  substances  are  likely  to  be  attacked  by  bacteria, 
and  what  changes  they  are  capable  of  producing  in  such  substances.  He 


2  PHARMACEUTICAL    BACTERIOLOGY. 

should  have  some  knowledge  of  the  effects  that  bacterially  deteriorated  sub- 
stances may  have  when  introduced  into  the  human  organism.  He  should  be 
qualified  to  sterilize  pharmaceuticals  as  is  now  required  in  the  pharmacopoe- 
ias of  several  foreign  countries  as  Austria,  Italy,  and  Belgium.  He  should 
know  something  of  the  comparative  value  of  the  numerous  disinfectants  and 
antiseptics  used  and  found  upon  the  market  and  should  know  how  to  stand- 
ardize these  agents  according  to  recent  bacteriological  methods.  The 
pharmacist  should  know  that  bacteria,  yeasts,  and  related  organisms  develop 
very  promptly  and  profusely  in  all  aromatic  waters;  in  carelessly  manipulated 
boiled  and  distilled  water;  in  dilute  solutions  of  all  acids  and  alkalies;  in 
dilute  alcohol  and  alcoholic  liquids;  tinctures;  infusions;  extracts,  solid  and 
liquid;  decoctions;  in  dilute  salt  solutions;  in  plant  juices;  mucilages;  emul- 
sions; elixirs;  wines;  in  syrups  of  all  kinds;  in  carelessly  manipulated  vege- 
table drugs,  crude  and  powdered;  in  drugs  from  the  animal  kingdom,  as 
ox-gall,  lard,  oils,  fats,  pepsin,  etc.  He  should  have  a  clear  comprehension 
of  antiseptics  as  germ  destroyers,  and  should  know  how  to  prepare  and  use 
them.  He  should  have  a  general  knowledge  of  alimentary  and  systemic 
phagocytosis;  of  leucocytosis  in  inflammatory  processes,  in  pus  formation, 
necrosis,  etc.  He  should  comprehend  immunity,  natural  and  acquired; 
he  should  know  about  opsonins  and  the  opsonic  index.  He  should  have  a 
general  knowledge  of  bacterial  enzymes;  of  toxins,  ptomaines,  leucomaines; 
of  antitoxins;  of  bacterial  vaccines.  He  should  have  a  special  knowledge  of 
the  source,  manufacture,  and  use  of  antitoxins  and  toxins,  modified  toxins, 
vaccine  virus,  and  related  products  used  in  medical  practice.  He  should  have 
a  general  knowledge  of  the  causation  of  the  more  common  bacterial  and 
protozoic  diseases.  He  should  have  special  instruction  in  the  disinfection 
of  public  and  private  dwellings,  and  should  be  able  to  cooperate  with  the 
physician  in  stamping  out  threatened  epidemics  and  in  carrying  out  public 
prophylactic  and  hygienic  measures.  To  attain  these  ends  a  knowledge 
of  bacteriology,  specialized  to  suit  the  needs  of  the  pharmacist,  is  absolutely 
essential. 

It  is  not  the  so-called  practical  side  of  bacteriology,  represented  by  dollars 
and  cents,  which  should  interest  the  pharmacist  in  this  science,  but  rather 
the  broader  view  of  his  profession  which  will  enable  him  to  perform  his 
duties  more  intelligently  and  more  efficiently.  The  man  whose  actions  are 
altogether  prompted  and  directed  by  the  dollar  sign  has  no  place  in  pharmacy 
or  in  medicine.  He  should  turn  to  some  non-professional  enterprise. 

As  yet  there  are  no  text-books  or  other  works  devoted  especially  to  phar- 
maceutical bacteriology.  Text-books  on  bacteriology  for  use  in  universities, 
medical  colleges,  and  technical  schools  are  not  suitable  for  use  in  colleges  of 
pharmacy.  Some  of  these  books  are  excellent  collateral  reading  for  phar- 
macists, but  most  of  them  are  of  such  a  highly  specialized  nature  that  they 


GENERAL   INTRODUCTION.  3 

would  no  doubt  do  more  harm  than  good  should  the  average  pharmacist 
attempt  to  use  them  as  a  practical  guide  in  the  performance  of  his  duties. 
Bacteriology  must  not  be  made  discouragingly  difficult  to  the  pharmacist,  in 
order  that  the  best  results  may  be  attained. 

Wherever  possible  the  college  instruction  in  pharmaceutical  _ bacteri- 
ology should  be  supplemented  by  visits  to  biological  laboratories  for  the 
manufacture  of  sera  and  bacterial  vaccines,  to  board  of  health  laboratories, 
quarantine  stations,  garbage  reduction  works,  etc.  Students  should  also  be 
assigned  special  reading.  Journals  and  special  treatises  on  bacteriology 
and  on  public  sanitation  should  be  consulted.  The  reports  on  bacteriological 
and  related  subjects  issued  from  time  to  time  by  the  United  States  Public 
Health  and  Marine  Hospital  Service  are  of  special  interest. 

The  following  references  are  given  for  the  benefit  of  those  students  who 
may  desire  further  information  regarding  the  earlier  conceptions  of  phar- 
maceutical bacteriology.  It  will  be  found  that  the  opinions  advanced  by  the 
authors  cited  differ  considerably. 

1.  Bacteriology   for   Pharmacists.     Pharm.    Journ.    Trans,,     23    (III), 
565,  865;  24  (III),  101,   1893. 

Largely  a  description  of  the  apparatus  employed  in  bacteriological  work, 
giving  special  attention  to  the  value  and  use  of  the  compound  microscope  in 
such  work. 

2.  H.  P.  Campbell.     Bacteria  Dangerous  to  Medicines.     Am.  Journ. 
Pharm.,  72,  113-118,     1890. 

3.  R.  G.  Eccles.     Pharmaceutical  Bacteriology.     Proc.  A.  Ph.  A.,  42, 
225-230,  1894. 

A  very  interesting  paper  on  the  theoretical  possibilities  of  pharmaceutical 
bacteriology. 

4.  J.  L.  Hatch.     Bacteriology.    Pharm.  Journ.  Trans.,  22   (III),  271, 

289>   33°>  l89J- 

A  series  of  lectures  delivered  before  the  alumni  association  of  the  Phila- 
delphia College  of  Pharmacy,  devoting  the  major  attention  to  the  morph- 
ology, physiology,  and  classification  of  bacteria. 

5.  R.   T.    Hewlett.     Bacteriology    in    its    Practical    Aspects.     Pharm. 
Journ.  Trans.,  25  (III),  819-820,  893-894,   1895. 

A  general  retrospect  of  bacteriology  as  a  possible  source  of  financial  gain 
to  the  pharmacist. 

6.  Smith  Ely  Jeliffe.     Moulds  and  Bacteria.  Druggists  Circular,   94-95, 
1897. 

A  description  of  some  of  the  more  common  moulds  and  bacteria  found 
medicinal  solutions.     Good  illustrations. 

7.  E.  Klein.     Bacteria,  Their  Nature  and  Function.     Pharm.  Journ. 
Trans.,  23  (III),  15,  35,   1893. 


4  PHARMACEUTICAL    BACTERIOLOGY. 

8.  W.  H.  Lymans.     Bacteriological  Culture  Apparatus.     Pharm.  Journ. 
Trans.,   1893.     (National  Druggist,  173,   1893.) 

9.  Albert  Schneider.     Pharmaceutical  Bacteriology.     Proc.  A.  Ph.  A., 
48,  186-189,  1894. 

10.  Specialism  in  Pharmacy,   begotten   by  Progress  in    Bacteriology. 
Pharm.  Journ.  Trans.,  25  (III),  625,   1895. 

Points  out  the  necessity  of  a  suitable  preparatory  training;  the  importance 
of  a  knowledge  of  the  use  of  antiseptics. 

11.  R.    Warrington.     The    Chemistry    of    Bacteria.     Pharm.    Journ. 
Trans.,  23  (III),  402,   1893.     (Pharm.  Era.,  104,  1894.) 


CHAPTER  II. 
HISTORICAL. 

It  must  be  evident  that  the  science  of  bacteriology  had  its  inception  with 
the  discovery  of  the  compound  microscope.  For  some  time  the  progress 
in  bacteriological  investigation  continued  parallel  with  the  progress  in  the 
mechanical  perfection  of  the  microscope  and  with  the  advance  in  microscop- 
ical technic.  Gradually,  however,  the  chemical  and  physiological  inve^ti- 
gations  pertaining  to  bacteria  gained  in  importance  and  significance.  Our 
knowledge  of  the  morphology  of  bacteria  as  revealed  through  the  compound 
microscope  has  been  practically  stationary  for  two  decades,  but  not  so  our 
knowledge  of  bacterial  products  and  bacterial  action.  The  methods  of 
bacteriological  technology  have  been  gradually  perfected,  and  the  prog- 
ress along  this  line  has  kept  pace  with  the  chemical  and  physiological 
investigations. 

Although,  as  indicated,  the  science  of  bacteriology  is  of  comparatively 
recent  origin,  yet  we  must  not  lose  sight  of  the  fact  that  many  of  the  ideas 
underlying  this  science,  as  now  comprehended,  were  advanced  in  remote  an- 
tiquity. For  this  reason  it  is  desirable  to  set  forth  these  earlier  concepts  in  a 
historical  review.  Most  of  the  writers  on  general  bacteriology,  who  make 
reference  to  the  history  of  the  subject,  almost  invariably  mention  the  older 
ideas  regarding  spontaneous  generation  as  being  the  forerunners  of  the  mod- 
ern ideas  of  bacteriology.  It  is,  however,  the  ancient  theories  and  beliefs 
pertaining  to  the  cause  of  decay,  disease,  and  epidemics  which  are  even  more 
directly  associated  with  the  first  more  important  discoveries  pertaining  to 
modem  bacteriological  pathology. 

For  the  purposes  of  simplification,  condensation,  and  greater  clearness 
the  historical  review  is  divided  into  periods  or  epochs.  It  is  not  possible, 
in  the  following  brief  outline,  to  cite  all  investigations  of  importance.  Only 
a  few  of  the  epoch-making  specialists  are  mentioned. 

Period  I. 

From  Hippocrates  (300  B.  C.)  to  Leeuwenhoek  (1656).  (The 
earliest  ideas  regarding  epidemics  and  spontaneous  generation.) 

From  the  earliest  times  the  more  scholarly  writers  mentioned  certain 
noxious  gaseous,  and  odoriferous  substances  or  erfluvias  as  being  the  cause 
of  epidemics.  These  effluvias  were  supposed  to  emanate  from  the  soil,  from 

5 


0  PHARMACEUTICAL   BACTERIOLOGY. 

the  air,  from  water,  stagnant  pools,  marshes,  from  decaying  and  putrescent 
substances,  from  crowded  habitations,  army  camps,  etc.  The  common 
people  throughout  the  world  and  throughout  all  ages  have  held  the  belief 
that  pestilence  and  disease  was  the  manifestation  of  divine  or  supernatural 
influence,  the  judgment  of  an  angry  deity,  a  punishment  inflicted  on  mankinti 
for  their  sins  and  iniquities,  beliefs  which  are  occasionally  asserted  even  at 
the  present  time.  Changes  of  season,  climatic  conditions,  arid  the  influence 
of  heavenly  bodies  were  also  considered  as  causative  of  diseases  of  an  epi- 
demic nature. 

/"'Animals,  such  as  rats,  mice,  and  insects,  have  long  been  recognized  as 
possible  carriers  of  disease.  An  English  investigator  has  recently  discovered 
some  very  excellent  sanitary  rules  in  the  Vedas  of  the  Hindus.  The  follow- 
ing is  a  translation  from  Book  VI,  verse  50,  of  the  Atharva-Veda. 

"Destroy  the  rat,  the  mole,  the  boring  beetle;  cut  off  their  heads,  O  asvins. 

"Bind  fast  their  mouths;  let  them  not  eat  our  barley;  so  guard  ye  twain  our  growing 
corn  from  danger. 

"  Hearken  to  me,  lord  of  the  female  borer,  lord  of  the  female  grub !  Ye  rough-toothed 
vermin. 

"Whate'er  ye  be,  dwelling  in  woods,  and  piercing,  we  crush  and  mangle  all  those 
piercing  insects." 

By  " piercing  insects"  no  doubt  mosquitos  are  meant.  If  the  injunctions 
were  literally  obeyed,  plague,  malaria,  and  certain  protozoic  diseases  would 
be  abolished  from  India. 

f-  Hippocrates  (460-377  B.  C.),  the  father  of  medicine,  considered  seasons 
and  winds  as  the  cause  of  pestilence,  particularly  the  long  continued  south- 
erly winds  (for  Greece),  and  a  warm,  humid,  clouded  atmosphere.  Galen 
(130-220  A.  D.)  held  similar  beliefs.  He  declared  that  diseases  arose  from 
a  putridity  of  the  air  or  from  atmospheric  and  weather  conditions.  Mar- 
cellinus  (359  A.  D.),  a  warrior  as  well  as  philosopher  and  historian,  declared 
that  the  decomposing  bodies  left  on  the  battlefield  were  the  cause  of  "  pesti- 
lential distempers,"  also  caused  by  extremes  in  weather,  by  marsh  effluvias, 
violent  heat,  and  a  vitiated  atmosphere.  Aetiusj^ fifth  century),  an  eminent 
physician,  declared  that  epidemics  or  common  diseases  were  caused  by  bad 
food,  bad  water,  immoderate  grief,  hunger,  excesses,  particularly  abundance 
following  extreme  want,  lack  of  exercise,  excessive  humidity,  and  putrid  sub- 
stances. Alpinus,  a  Venetian  physician  of  the  sixteenth  century,  explained 
how  the  cause  of  plagues  and  epidemics  may  be  carried  by  persons  or  in 
cargoes.  He  pointed  out  that  a  given '  disease  from  one  country  is  more 
malignant  than  the  same  disease  from  another  country.  During  the  dark 
and  middle  ages  various  ecclesiastical  and  lay  writers  ascribed  epidemics  and 
pestilence  to  a  variety  of  causes — the  wrath  of  God,  to  demons  or  evil  spirits, 
comets,  meteors,  earthquakes,  volcanic  eruptions,  cyclones,  eclipses  of  the 
sun,  terrific  storms,  wars,  famines,  great  fires,  etcl  Even  as  late  as  1799 


HISTORICAL.  7 

no  less  an  authority  than  Noah  Webster  makes  the  following  declaration: 
"All  the  great  plagues  which  have  afflicted  mankind  have  been  accompanied 
with  violent  agitations  of  the  elements.  The  phenomenon  most  generally 
and  closely  connected  with  pestilence  is  an  earthquake.  From  all  the  facts 
which  I  can  find  in  history,  I  question  whether  an  instance  of  any  considerable 
plague,  in  any  country,  can  be  mentioned  which  has  not  been  immediately 
preceded  by,  or  accompanied  with,  convulsions  of  the  earth.  If  any  excep- 
tions have  occurred,  they  have  escaped  my  researches.  It  does  not  happen 
that  every  place  where  pestilence  prevails  is  shaken;  but  during  the  progress 
of  the  disease  which  I  denominate  pestilence,  and  which  runs,  in  certain 
periods,  over  large  portions  of  the  globe,  some  parts  of  the  earth,  and  especi- 
ally those  which  abound  most  with  subterranean  fire,  are  violently  agitated." 
Were  Noah  Webster  alive,  he  would  certainly  cite  the  recent  plague  on  the 
Pacific  Coast  as  bearing  out  his  assertions.  On  April  18,  1906,  the  coast 
region  about  San  Francisco  was  certainly  "violently  agitated,"  and  this  phe- 
nomenon was  followed  by  the  plague  (black  pest,  bubonic  plague).  But 
what  were  the  actual  facts?  The  plague  had,  in  all  probability,  existed  in  a 
sporadic  form  in  "Chinatown,"  in  San  Francisco,  and  in  other  places  on  the 
Pacific  coast  for  many  years.  In  1903  several  authentic  cases  came  to  notice 
and  were  reported.  The  reasons  why  the  disease  had  not  previously  gained 
a  stronger  foothold  in  San  Francisco  are  several.  Chinatown  is  more  or  less 
isolated  (socially,  at  least)  from  the  rest  of  the  city,  and  the  poorer,  more 
filthy  class  of  the  Chinese  do  not  as  a  rule  mingle  with  the  white  population. 
The  disease  is  an  Oriental  filth  disease.  After  the  earthquake  and  fire  of 
April  18-22,  1906,  the  Chinese  of  all  classes,  the  plague-infected  rats  and 
fleas  of  the  Chinese  quarters,  became  thoroughly  intermingled  with  the 
rest  of  the  stricken  population,  and  as  a  result  there  were  established  several 
new  foci  of  plague  infection,  which  accounted  for  the  increase  in  plague 
cases  in  1907,  a  condition  which  was  soon  under  control,  thanks  to  the 
strenuous  efforts  of  the  federal  government,  the  board  of  health,  and  various 
citizens'  organizations 

Several  writers  of  remote  times,  as  well  as  occasional  writers  of  the  dark 
and  middle  ages,  held  the  opinion  that  the  cause  of  disease,  the  disease- 
producing  effluvias,  might  be  carried  long  distances  by  air  currents,  in  ships, 
or  by  caravans,  and  that  the  poison  may  enter  the  system  via  the  air  pas- 
sages, through  the  skin,  or  through  the  digestive  tract.  Hodges,  an  English-  . 
man,  who  wrote  a  treatise  on  the  London  plague  of  1665,  declared  that  some 
essential  alteration  in  the  air  is  necessary  to  the  propagation  of  this  disease. 
That  is,  the  "  nitro-aerial "  principle,  which  causes  or  invigorates  plant  and 
animal  life,  is  supposed  to  become  vitiated. 

The  corrupting  principle  is  a  "subtle  aura  or  vapor"  which  is  "extricated 
from  the  bowels  of  the  earth."     This  plague-causing  poison  was  said  to 


8 


PHARMACEUTICAL    BACTERIOLOGY. 


affect  trees  and  other  plants,  fishes  and  other  animals,  as  well  as  man.  Dr. 
Mead  declared  that  epidemics  were  caused  by  (i)  diseased  persons,  (2) 
goods  imported  from  infected  places,  and  (3)  a  vitiated  or  poisoned  state  of 
the  air,  notions  which  may  be  considered  as  the  direct  forerunners  of  the 
germ  theory  of  disease. 

Let  us  now  go  back  and  consider  the  ancient  ideas  regarding  spontane- 
ous generation.  Anaximander,  of  Miletus,  who  lived  during  the  forty- 
third  Olympiad  (610  B.  C.),  believed  that  many  animals  developed  de  novo, 
from  moisture  and  water  acted  upon  by^sun  and  warmth.  The  extremist, 
Empedodes  of  Agrigentum  (450  B.  C.),  declared  that  all  living  things  upon 
the  earth  were  capable  of  originating  spontaneously.  Aristotle  (384  B.  C.) 
taught  that  some  plants  and  animals  originated  spontaneously.  Ovid,  some 


FIG.  i. — From  the  Arcana  Natures  of  A.  van  Leeuwenhoek.  The  first  published 
illustration  of  bacteria.  These  bacilli  of  the  mouth  cavity  were  seen  with  the  aid  of  simple 
lenses  only,  a,  b,  bacilli;  c,  a  spirillum;  e,  perhaps  chain  forms  of  bacilli;  d.  illustrating 
the  characteristic  motion  of  certain  bacilli  (n  to  m). 

three  centuries  later,  gives  instructions  how  to  create  bees  spontaneously  in 
the  carcasses  of  horses.  To  within  recent  times  the  belief  that  certain 
animals  could  originate  spontaneously,  that  is,  without  a  pre-existing  parent, 
was  quite  general,  and  differed  only  in  grotesqueness.  Cardan  as  late  as 
1542  declared  that  water  created  fishes,  and  that  many  fermentative  proc- 
esses created  animals.  Van  Helmont  gives  instructions  how  to  produce  mice 
artificially.  Kircher  boldly  declared  that  he  had  seen  certain  animals 
develop  spontaneously  before  his  eyes.  Paracelsus  gives  instructions  how  to 
make  homunculi.  The  instructions  are  quite  simple.  Certain  substances 
are  placed  in  a  bottle,  the  bottle  is  well  stoppered  and  buried  in  a  manure 
heap.  Every  day  certain  incantations  must  be  pronounced  over  the  bottle 
in  the  manure  heap.  In  time,  Paracelsus  declared,  a  small  living  human 
being  (homunculus)  will  appear  in  the  bottle.  Paracelsus,  however,  naively 


HISTORICAL.  9 

admits  that  he  has  never  succeeded  in  inducing  the  homunculus  to  continue 
alive  after  being  taken  from  the  bottle.  Gradually  these  grotesque  and 
extreme  opinions  regarding  spontaneous  generation  were  abandoned,  and  it 
was  declared  that  only  the  lower  plants  and  animals,  such  as  seaweeds,  algae, 
lichens,  lice,  mites,  maggots,  etc.,  could  develop  spontaneously.  -In  fact, 
we  can  find  fairly  intelligent  individuals  to-day  who  firmly  believe  that 
certain  animals,  as  lice,  mites,  etc.,  can  originate  without  a  parent,  and  that 
the  hair  from  the  tail  or  mane  of  a  horse  will  change  into  a  worm  or  snake  if 
placed  in  a  bottle  of  water  and  exposed  to  light  and  warmth. 

From  the  earliest  records  we  learn  that  the  value  of  disinfectants  in  pre- 
venting the  spread  of  infectious  diseases  (epidemics  and  plagues)  was  known. 
Ovid  states  that  the  shepherds  of  his  time  used  burning  sulphur  for  bleach- 
ing wool  and  to  free  it  from  infectious  diseases.  In  times  of  plagues,  big 
fires  were  made  to  stay  the  ravages  of  pestilential  diseases.  The  Mosaic 
law  is  replete  with  instructions  regarding  cleanliness  as  a  means  of  pre- 
venting disease.  Wine  was  highly  valued  as  a  dressing  for  wounds,  having 
the  effect  of  preventing  or  checking  pus  formation.  ) 

Period  II. 

From  Leeuwenhoek  (1656)  to  Schwann  (1837).  (Discovery  of 
micro-organisms  and  the  early  investigations  regarding  their 
activities.) 

As  early  as  1646  Kircher  suggested  that  certain  diseases  might  be  due 
toj£ery_jnjnutfi  organisms  which  were  supposed  to  originate  spontaneously 


FIG.  2. — From  Arcana  Naturae.    Cell  structure  of  cork.   Cell-lumen  is  shaded  and  cell-walls 

are  shown  light. 

under  certain  conditions.  Anton  van  Leeuwenhoek  is  very  justly  called  the 
father  of  microscopy,  and  to  him  must  undeniably  be  given  the  credit  of 
first  having  discovered  and  actually  figured  microbes  and  other  micro- 
organisms. His  Arcana  Nature  was  published  in  1656  in  four  volumes. 
It  is  a  most  interesting  work,  and  contains  many  good  illustrations  showing 


10  PHARMACEUTICAL   BACTERIOLOGY. 

microbes  of  the  mouth  cavity,  infusoria  of  stagnant  water  and  cellular 
structure  of  vegetable  tissues.  He  observed  the  motion  of  bacteria  and  in- 
fusoria, made  measurements,  illustrated  capillary  circulation  in  the  web 
of  the  frog's  foot,  etc.  He  was  closely  followed  by  Robert  Hooke,  who 
published  his  Micrographia  in  1658.  The  discoveries  of  Leeuwenhoek 
and  Hooke  were  certainly  epoch-making.  A  new  world  of  minute  organisms 
was  made  known,  the  question  of  spontaneous  generation  received  a  new 
turn,  and  the  way  to  the  discovery  of  the  causes  of  disease  and  fermentation 
was  paved.  In  1660  Leeuwenhoek  discovered  yeast  cells.  From  1660  to 
1760  the  microscope  was  actively  employed  by  a  few  investigators,  and  addi- 
tions were  slowly  made  to  the  list  of  micro-organisms.  Audry  (1701)  desig- 
nated microbes  worms.  Muller,  of  Copenhagen  (1786),  grouped  them 
under  two  divisions,  monas  and  vibrio.  In  1743  Henry  Baker,  of  England, 
published  his  work,  "The  Microscope  Made  Easy,"  from  which  it  would 
appear  that  very  little  progress  had  been  made  since  the  time  of  Leeuwen- 
hoek (1656). 

As  early  as  1686  Franceso  Redi  doubted  that  maggots  were  generated 
de  novo  in  putrid  meats.  He  noticed  that  the  presence  of  the  maggots  was 
preceded  by  swarms  of  flies  which,  he  concluded,  had  something  to  do  with 
the  development  of  the  maggots.  He  found  that  meat  from  which  the  flies 
were  excluded  by  means  of  paper  or  a  very  fine  mesh  wire  screen,  simply 
decayed  without  any  development  of  maggots.  The  paper  cover  and  the  fine 
screen  kept  the  eggs  of  the  flies  from  being  deposited  on  the  meat,  and  the 
meat  was  not  infested  by  maggots,  which,  as  Redi  rightly  conjectured, 
developed  from  the  eggs  of  the  fly-like  imago.  This  very  simple  but  reli- 
able experiment  did  much  to  create  doubt  as  regards  the  correctness  of  the 
theory  of  spontaneous  generation  and  other  related  beliefs. 

Spallanzani  (1777)  was  among  the  first  to  demonstrate  experimentally 
that  boiling  and  hermetically  enclosing  fermentable  liquids  prevented  fer- 
mentation. Ehrenberg  (1828)  discovered  microscopic  organisms  in  dust 
and  in  water,  and  in  1833  he  classified  all  known  bacteria  under  four  orders, 
bacterium,  vibrio,  spirillum,  and  spirocheta.  Cagniard-Latour  and  von 
Schwann  (1836)  discovered  the  vegetable  nature  of  yeast,  and  in  1837 
Schwann  decleared  that  yeast  was  the  direct  cause  of  fermentative  changes 
resulting  in  the  liberation  of  alcohol  and  CO2,  and  that  the  causes  of  decay 
were  to  be  found  in  the  atmosphere.  Berzelius  (1827)  declared  that  the  yeast 
cells  were  the  direct  cause  of  fermentation.  F.  Schulze  (1836)  prevented 
decay  in  liquids  containing  certain  organic  substances  by  first  heating  or 
boiling  them  and  excluding  the  air  by  means  of  a  layer  of  oil  or  by  closing 
the  container  with  cotton  and  supplying  it  with  air  which  had  been  ster- 
ilized by  passing  through  sulphuric  acid.  Braconnot  (1831)  advanced  the 
theory  that  yeast  cells  had  the  power  of  holding,  and  condensing  within  the 


HISTORICAL. 


II 


cell-substance,  the  oxygen  of  the  air  and  conducting  it  to  the  substances 
undergoing  fermentation,  resulting  in  the  splitting  up  of  sugar  into  alcohol 
and  carbonic  acid  gas. 

The  question  of  spontaneous  generation  was  again  discussed  with  renewed 
energy.  The  belief  that  larger  animals  could  originate  de  novo  was  quite 
generally  abandoned,  but  it  was  very  persistently  argued  that  micro-organisms, 
maggots  and  a  few  other  very  small  animals  could  thus  develop.  Bastian 
was  perhaps  the  leader  in  the  arguments  in  favor  of  spontaneous  generation, 
opposed  by  Schwann,  Pasteur,  and  others.  Schroeder  and  von  Dusch  dem- 
onstrated that  decay  could  be  prevented  by  boiling  and  supplying  air  that 
had  been  filtered  through  cotton.  Pasteur  (1862)  used  bent  tubes  to  supply 
air  to  the  previously  sterilized  (by  heating)  substance,  as  shown  in  Fig.  3. 


FIG.  3. — Flask,  containing  an  organic  substance,  a,  hermetically  closed  by  means 
of  a  stopper,  b.  The  bent  tube  is  open  at  e,  admitting  air.  Dust  and  microbes  lodge 
at  the  bends  d  and  c. 

The  microbes  in  the  air  passing  through  the  tube  are  deposited  (by  gravity) 
in  the  lower  bends  of  the  tube.  Those  favoring  the  theory  of  spontaneous 
generation  nevertheless  continued  their  arguments.  It  was  pointed  out  that 
changes  of  decay  took  place  in  eggs,  in  internal  tissues  and  organs  of  the 
dead  as  well  as  in  the  living,  etc.,  where,  it  was  supposed,  microbes  could 
not  possibly  have  access.  However,  further  convincing  experiments 
gradually  silenced  all  opposition.  Bastian  and  a  few  followers  took  practi- 
cally their  last  stand  in  1875,  an^  smce  tnat  time  no  scientist  of  repute  has 
ever  argued  in  favor  of  spontaneous  generation,  though  the  question  of  the 
primal  origin  of  living  things  remains  unanswered. 

Vaccination  as  a  protection  against  virulent  small-pox  was  practised 
early  in  the  eighteenth  century  in  Turkey  and  other  Oriental  countries,  and 
was  introduced  into  Europe  via  England  through  the  influence  of  Lady 
Mary  Wortley  Montagu.  A.  von  Humboldt  states  that  the  Mexicans 
practised  vaccination  at  a  very  early  period.  This  early  vaccination  mate- 


12  PHARMACEUTICAL    BACTERIOLOGY. 

rial  was  obtained  from  a  pustule  of  a  small-pox  patient,  and  not  from  the  cow, 
as  at  present.  The  immunity  against  subsequent  attacks  was  established, 
but  the  disease  transmitted  through  this  older  method  of  vaccination  was 
severe  and  often  fatal;  besides,  the  general  vaccination  was  a  source  of 
spreading  the  disease.  In  1840  this  form  of  vaccination  was  prohibited 
in  England  by  act  of  Parliament. 

In  1768  Jenner's  attention  was  attracted  to  the  value  of  vaccination,  and 
after  a  series  of  patient  researches  he  perfected  the  method  of  vaccination 
by  means  of  the  virus  obtained  from  a  cow  which  had  been  inoculated  with 
small-pox  (vaccinia).  Jenner  established  the  first  public  institution  for 
vaccination  in  1799,  and  in  the  following  year  the  practice  was  introduced 
into  France,  Germany,  and  the  United  States.  Vaccination  writh  vaccinia 
material  is  now  universal  in  all  civilized  countries  and  in  countries  under 
civilized  control,  and  as  a  result  small-pox  in  an  epidemic  form  does  not  occur 
in  these  countries,  and  the  disease  has  become  less  and  less  virulent,  so  that 
it  is  no  longer  the  dreaded  scourge  that  it  was  two  centuries  ago.  In  spite 
of  the  beneficent  influence  of  vaccination,  there  are  individuals  who  oppose 
this  simple,  harmless  operation  with  all  the  energy  that  ignorance  is  capable 
of.  Civilized  countries  are  beginning  to  raise  the  long-enforced  small-pox 
quarantine  as  a  wholly  unnecessary  infliction,  because  vaccination  makes  the 
spreading  of  small-pox  impossible.  France  has  raised  the  quarantine, 
and  so  have  several  other  countries,  examples  which  will  no  doubt  soon  be 
followed  generally.  In  conclusion,  it  is  of  interest  to  note  that  the  primary 
cause  of  small-pox  is  unknown  even  to  this  day.  No  organism  has  thus 
far  been  isolated  from  diseased  tissues  to  which  small-pox  manifestations 
could  be  ascribed. 

Period  III. 

r 

From  Schwann  (1837)  to  Pasteur  (1862).  (Investigations  per- 
taining to  the  relationship  of  micro-organisms  to  fermentation  and 
disease.) 

The  discoveries  of  the  cause  of  fermentation,  of  decay,  and  of  wound 
infection  are  closely  associated.  Many  centuries  ago  Varro  expressed  it  as 
his  opinion  that  certain  minute  animals,  breeding  in  marshy  places,  got  into 
the  system  through  mouth  and  nostrils  and  caused  the  disease  and  decay  of 
tissues.  Theodoric  (1260)  taught  that  wound  infection  came  from  the  air. 
To  prevent  such  infection  he  applied  wine,  which  is  known  to  be  somewhat 
antiseptic.  John  Colbach  (1704)  described  a  "new  and  secret  method  of 
treating  wounds  by  which  healing  took  place  without  inflammation  or 
suppuration." 

From  earliest  time  up  to  as  late  as  1860,  it  was  quite  generally  taught 


HISTORICAL.  13 

that  all  normal  healing  of  wounds  and  cuts  must  be  preceded  by  pus-for- 
mation. A  "laudable  pus"  was  recognized,  the  presence  of  which  was 
looked  upon  as  a  hopeful  sign  and  indicated  that  repair  was  proceeding 
favorably.  If  the  laudable  pus  which  was  of  a  whitish  creamy  consistency 
changed  to  a  watery  consistency,  it  was  considered  an  unfavorable  sign. 

After  Schwann  and  others  had  demonstrated  that  fermentation  was  due 
to  the  presence  of  yeast  cells,  and  it  was  proven  conclusively  that  decay  was 
caused  by  bacteria,  the  relationship  of  bacteria  to  disease  began  to  receive 
consideration.  Rayner  and  Devaine  (1850)  found  bacterial  rods  in  animals 
suffering  from  splenic  fever.  As  early  as  1840  Henle,  who  is  by  some  con- 
sidered the  father  of  modern  bacteriology,  made  some  very  valuable  deduc- 
tions regarding  the  relationship  of  micro-organisms  to  disease.  He  recog- 
nized a  "contagium"  (the  active  cause  of  the  disease  associated  with  micro- 
organisms), which  was  supposed  to  be  air-like  and  yet  at  the  same  time 
fixed.  It  was  supposed  to  retain  its  activity  for  years  in  the  dry  state.  An 
unweighable  and  unmeasurable  quantity  of  this  substance  may  cause  an 
extensive  epidemic.  Air  currents  can  carry  the  contagium  great  distances 
and  cause  epidemics  in  widely  separated  areas.  Bassi  (1835)  declared  that 
a  fungus  was  the  cause  of  the  muscardine  disease  of  silkworms.  Pollender 
(1855)  reported  that  bacteria  caused  anthrax,  verified  by  Devaine  in  1863. 
Hallier,  an  enthusiast  but  not  reliable  as  an  investigator,  declared  that 
scarlet  fever,  measles,  typhus,  and  cholera  were  caused  by  bacteria.  His 
deductions  were,  however,  not  based  upon  scientific  research  and  proof. 
Rindfleisch  (1866)  and  Waldeyer  (1868)  gave  considerable  attention  to 
wound  infection,  which,  they  declared,  was  due  to  microbic  invasion.  In 
i86o_Pasteur  demonstrated  the  microbic  cause  of  the  silkworm  disease 
which  interfered  very  seriously  with  the  silk  industry  in  France.  Pasteur 
and  Klebs  demonstrated  experimentally  that  bacteria  could  be  grown  in 
artificial  culture  media,  and  Robert  Koch  proved  that  the  pathogenic  mi- 
crobes actually  secreted  the  disease-causing  substance.  This  was  demon- 
strated by  transferring  an  infinitely  small  quantity  of  the  germ  material  from 
a  diseased  organ  to  a  suitable  culture  medium  and  making  sub-cultures, 
until  the  last  culture  must  contain  less  than  the  trillionth  part  of  the  original 
substance.  Nevertheless,  inoculations  from  the  last  culture  developed  the 
disease  with  full  energy.  This  experiment  was  made  to  meet  the  assertions 
that  the  cause  of  the  disease  did  not  reside  in  the  bacterium,  and  that  the 
bacterium,  if  present  in  the  disease,  was  merely  incidental  to  and  not  causa- 
tive of  the  disorder. 

A  heated  controversy  continued  for  some  time.  Such  authorities  as 
Liebig,  Na'geli,  Bastian,  Cohrij  Billroth,  Hiller,  Schroeder,  Hoppe-Seyler, 
Kiihne,  Tiegel,  Sanderson,  Nencki,  Serval,  and  Paschutin  declared  that 
micro-organisms  were  not  the  cause  of  decay,  fermentation,  and  disease; 


14  PHARMACEUTICAL    BACTERIOLOGY. 

that  these  changes  were  due  to  chemical  substances.  However,  such  men 
as  Pasteur,  Koch,  Panum,  Klebs,  and  others  forged  link  after  link  in  the 
chain  of  evidence  connecting  the  causative  relationship  of  bacteria  to  disease. 

Period  IV. 

From  Pasteur  (1862)  to  Behring  (1890).  (Period  of  remarkable 
activity  in  pathological  bacteriology.) 

It  would  be  impossible  in  a  brief  review  to  cite  all  of  the  important 
investigations  of  this  period.  Pasteur,  Koch,  and  others  had  already  given 
the  subject  of  bacteriological  technic  considerable  attention.  The  most 
suitable  culture  media,  laboratory  apparatus,  stains,  etc.,  were  determined. 
The  compound  microscope  had  now  reached  a  high  degree  of  perfection,  and 
the  oil-immersion  lenses  made  the  closer  study  of  the  morphology  of  bacteria 
possible. 

As  might  be  expected,  the  importance  of  germicides  irr^urgery  received 
first  attention.  The  "laudable  pus-"  formation  ideas  were  at^andoned.  It 
became  the  surgeon's  duty  to  induce  " primary  union"  or  healing  by  " first 
intention,"  that  is,  healing  without  any  pus  formation  whatever.  This 
demanded  that  the  surfaces  of  the  incision  be  brought  in  close  contact,  and 
that  all  bacterial  infection  be  prevented  by  the  use  of  antiseptic  dressings, 
antiseptic  solutions  in  the  form  of  irrigations  and  sprayings,  etc.  Sir  Joseph 
Lister,  of  Scotland  (1875),  brought  the  use  df  disinfectants  in  surgery  to  a 
high  degree  of  perfection,  and  modern  antiseptic  surgery  is  often  designated 
"Listerism."  The  modern  proprietary  antiseptic  "listerine"  is  named 
after  this  eminent  surgeon.  The  chief  antiseptic  of  Lister  and  his  followers 
was  carbolic  acid,  which  was  used  for  free  wound  irrigation  and  general 
disinfection.  He  operated  in  a  spray  of  carbolic  acid  solution.  As  late  as 
1890  there  was  to  be  found  an  occasional  lecturer  in  a  college  of  medicine 
who  held  out  against  the  germ  theory,  and  not  a  small  number  of  the  eminent 
opponents  mentioned  in  the  previous  period  carried  their  mistaken  notions 
with  them  to  the  grave. 

The  name  of  Robert  Koch  will  stand  throughout  the  ages  as  the  leader  in 
modern  bacteriological  science.  Early  in  life  he  was  convinced  of  the  cor- 
rectness of  the  germ  theory  of  disease,  but  his  first  contributions  to  bacterio- 
logical'science  awakened  a  storm  of  opposition.  Billroth,  of  Vienna,  and 
others  persisted  in  declaring  that  microbes  were  not  causative  of  pus-forma- 
tion or  of  the  development  of  disease;  but  that  microbes  might  be  accident- 
ally present,  due  to  the  action  of  a  "  phlogistic  zymoid"  which  developed  in 
the  animal  organism. 

In  1882  the  French  government  sent  a  medical  commission  to  India  to 
determine  if  possible  the  cause  of  Asiatic  cholera,  but  the  commission  re- 


HISTORICAL.  15 

turned  with  a  negative  report  as  far  as  a  bacterial  cause  of  the  disease  was 
concerned.  In  1883  the  German  government  sent  a  similar  commission, 
headed  by  Robert  Koch,  and  the  report  of  this  commission  was  that  Asiatic 
cholera  was  caused  by  a  bacillus,  the  famous  comma  bacillus  of  Koch.  The 
work  of  Koch  in  connection  with  the  study  of  cholera  seemed  to  act  as  a 
wonderful  stimulus,  and  other  eminent  investigators  made  important  dis- 
coveries within  the  year  or  two  following.  Klebs  and  Loffler  discovered 
the  diphtheria  bacillus  in  1884.  Fraenkel,  Weichselbaum,  and  Friedlander 
discovered  the  pneumococcus  in  1884.  Nicolaier  and  Kitasato  discovered 
the  tetanus  bacillus  in  1884.  Loffler  and  Schiitz  discovered  the  glanders 
bacillus  in  1882,  and  the  bacillus  of  hog  erysipelas  (Rothlauf)  in  1885. 

Pasteur  in  1881  made  his  first  experiments  in  reproducing  rabies  in 
susceptible  animals  by  inoculation  with  material  obtained  from  the  spinal 
cord,  medulla  oblongata,  and  lobes  of  the  brain  o£  animals  dead  from 
rabies.  In  1884  he  reported  his  experiments  pertaining  to  the  modification 
of  the  virulence  of  rabies  by  successive  inoculations  into  susceptible  animals. 
His  use  of  this  modified  rabies  virus  as  a  means  of  preventing  a  severe  and 
fatal  course  of  the  disease  in  those  bitten  by  animals  suffering  from  hydro- 
phobia, is  familiar  to  all.  Thousands  of  cases  have  been  treated  successfully 
at  Pasteur  institutes  established  throughout  the  larger  cities  of  the  civilized 
world. 

The  above  are  only  a  few  of  the  important  investigations  of  this  period. 
The  causative  relationship  of  microbes  to  certain  diseases  was  undeniably 
established.  The  voices  of  opposition  were  silenced. 

This    period    is    especially    nntahlp    fpr    the    dpv^npmppt    nf    flntisppfir 

surgery.  As  a  result,  operations  were  no  longer  dreaded  as  in  former  times. 
Fatal  infections  following  operations  now  became  rare.  Thousands  of  lives 
are  saved.  To  remove  or  destroy  the  pus  germs  in  open  wounds  or  to  pre- 
vent the  access  of  germs  to  wounds,  cuts,  and  abrasions,  has  become  a  simple 
matter,  a  simple  mechanical  application  of  suitable  antiseptics. 

The  progress  of  purely  medical  bacteriology  was  not  so  marked.  Al- 
though it  was  proven  that  certain  diseases  were  due  to  bacteria,  there  were 
no  satisfactory  means  of  destroying  them  in  the  system.  Internal  antiseptics 
were  tried,  but  without  satisfactory  results,  as  a  rule.  However,  preventive 
medicine  based  on  a  bacteriological  knowledge  gave  good  results. 

Period  V. 

From  Behring  (1890)  to  Wright  (1907).  (Discovery  of  serum 
therapy,  bacterial  vaccines,  and  development  of  utilitarian  bac- 
teriology.) 

The  subject  of  immunity  from  disease  received  early  attention.     Age 


1 6  PHARMACEUTICAL   BACTERIOLOGY. 

immunity,  race  immunity,  animal  immunity,  individual  immunity,  artificial 
immunity,  natural  immunity,  acquired  immunity,  etc.,  attracted  attention 
and  received  careful  consideration.  Metchnikoff  (1884)  explained  im- 
munity on  the  supposition  that  certain  white  corpuscles  (leucocytes,  phago- 
cytes) of  the  blood  devoured  the  microbes  which  entered  the  system.  These 
white  blood  corpuscles  are  the  guardians  of  health.  They  attack  and  feed 
upon  any  disease  germs  which  may  enter  the  body,  either  via  the  digestive 
tract,  the  respiratory  tract,  or  via  the  circulatory  system.  If  the  leucocytes 
are  deficient  in  number,  or  if  the  microbes  are  excessive  in  number,  disease 
will  develop.  This  theory  had  numerous  followers,  as  well  as  opponents. 
It  is  now  generally  accepted  as  correct,  borne  out  by  observation  and  by 
experimental  evidence. 

The  next  important  discovery  was  that  blood  serum  had  bactericidal  prop- 
erties in  a  varying  degree,  and  that  in- addition  to  this  there  was  something 
in  the  blood  which  had  a  tendency  to  neutralize  or  destroy  the  action  of  the 
poisons  or  toxins  formed  by  pathogenic  .microbes.  No  one  particular 
bacteriologist  can  be  said  to  have  made  these  discoveries.  We  can  only 
name  a  few  of  the  leading  investigators  who  worked  along  these  lines,  leading 
to  the  discovery  of  the  relationship  of  immunity  and  antitoxins — Behring, 
Brieger,  Buchner,  Calmette,  Chamberland,  Ehrlich,  Emmerich,  Fliigge, 
Frankel,  Hiieppe,  Jetter,  Kitasato,  Klemperer,  LofBer,  Rankin,  Roux, 
Wassermann,  and  others.  These  eminent  authorities  have  demonstrated 
the  possibility  of  developing  or  aiding  the  antitoxic  or  immunizing  power 
of  the  blood  or  of  the  body  cells  by  introducing  sera  obtained  from  the  blood 
of  animals  in  which  the  antitoxic  power  is  naturally  high  or  is  made  so  as 
the  result  of  special  treatment.  Numerous  sera  (containing  antitoxins  and 
toxins)  were  tried;  the  one  which  first  proved  entirely  satisfactory  was  the 
diphtheria  antitoxin  of  Behring,  which  is  now  in  universal  use.  O.thers 
are  used  more  or  less  successfully,  and  some  are  still  in  the  experimental 
stage. 

In  1890  Koch  reported  on  a  "lymph"  to  be  used  in  the  treatment  of 
tuberculosis.  This  lymph  was  a  glycerin  extract  of  the  toxin  of  the  bacillus 
of  tuberculosis,  and  was  to  be  used  in  the  treatment  of  this  dread  disease,  but 
the  hopes  of  Koch  were  not  realized,  as  the  remedy  proved  a  failure,  and  it 
soon  fell  into  disuse,  to  be  again  taken  up  very  recently.  In  1907  Wright 
made  known  his  discovery  of  opsonins.  According  to  this  authority,  there 
exist  in  the  blood  certain  substances  which  have  the  power  of  acting  on  the 
invading  bacteria  in  such  a  manner  as  to  render  them  more  liable  to  be 
attacked  and  assimilated  by  the  white  blood-corpuscles  or  leucocytes.  There 
are  possibly  as  many  opsonins  as  there  are  microbes  capable  of  being  di- 
gested by  the  leucocytes.  The  microbe-devouring  power  of  the  leucocytes 
can  be  increased  by  the  use  of  bacterial  vaccines,  which  consist  of  suspensions 


HISTORICAL.  17 

of  microbes.     Very  minute  quantities  are  injected  into  the  system,  and  the 
resulting  reaction  increases  the  power  referred  to. 

Toxins  of  bacterial  origin  received  the  attention  of  investigators,  and 
antibodies  (antitoxins)  were  extensively  discussed  as  to  their  possible  relation- 
ship to  health  and  disease.  Enzymes,  in  their  relationship  to  life 
processes  in  plants  and  in  animals,  were  investigated.  It  is  now  supposed 
that  soil  toxins  of  plant  origin,  as  well  as  those  of  bacterial  origin,  influence 
plant  growth.  Glandular  preparations  (ductless  glands)  have  been  care- 
fully tested,  and  several  of  these  are  in  use. 

As  the  result  of  Wright's  discovery  of  the  use  of  bacterial  vaccines  in 
increasing  the  opsonic  index,  the  tuberculin  (lymph)  of  Koch  was  again 
tried  in  the  treatment  of  tuberculosis,  apparently  with  some  success. 

It  was  found  that  there  were  many  bacteria  other  than  those  which 
caused  disease  in  animals  and  plants.  Some  were  found  to  be  decidedly 
beneficial.  Bacterial  cultures  were  employed  in  butter-making  (ripening  of 
cream),  in  cheese-making,  in  tanning,  in  paper-making,  siloing,  etc.  Some 
bacteria  are  employed  to  exterminate  certain  pest  animals.  A  microbic 
chintz  bug  exterminator  was  tried  in  1895-97,  but  it  proved  a  failure. 
Microbic  rat  and  mice  exterminators  (azoa,  ratite,  mouratus,  etc.)  are  now 
being  tested,  and  they  appear  to  be  quite  successful,  at  least  in  certain 
localities  and  under  certain  conditions.  A  microbic  rabbit  exterminator 
has  been  tried  in  Australia. 

In  1879  Dr.  Frank,  of  Berlin,  began  his  investigations  of  the  leguminous 
root  nodule  microbes.  In  1893  the  writer  attempted  to  utilize  these  microbes 
in  increasing  the  yield  of  certain  gramineous  crops.  In  1896  Nobbe  and 
Hiltner,  of  Germany,  introduced  a  patented  microbic  fertilizer  for  legumin- 
ous plants.  In  1907  a  California  soil  microbe  was  isolated  which  appears 
to  be  especially  active  in  promoting  the  growth  of  sugar  beets.  This  experi- 
ment led  to  the  supposition  that  perhaps  every  species  of  plant  has  its'  pecu- 
liar bacterial  flora,  symbiotically  (mutually  beneficent)  associated  with  the 
root  system,  mutually  essential  to  active  development.  The  importance  of 
soil  bacteria  in  setting  free  plant  foods  has  been  demonstrated  by  numerous 
investigators  of  Europe  and  of  the  United  States.  Yeast  and  mould  organ- 
isms are  practically  utilised  in  the  manufacture  of  beer,  sake,  and  other 
food  and  drink  products. 

outline  of  the  history  of  bacteriology  may  be 


The  above   condensed 


summed  up  as  follows: 

1.  Ancient  conceptions  of  disease  and  of  spontaneous  generation,  dating 
back  to  500  years  B.C. 

2.  Discovery  of  micro-organisms  about  1660  by  Leeuwenhoek,  followed 
by  the  work  of  Robert  Hooke  and  a  few  others. 

3.  Discovery  of  bacteria  in  air,  dust,  and  decaying  substances,  and  the 

2 


1 8  PHARMACEUTICAL   BACTERIOLOGY. 

causal  relationship  of  microbes  to  decay,  and  of  the  yeast  organisms  to 
fermentation. 

4.  Disproving  the  theory  of  spontaneous  generation,  by  Schwann  and 
others,  about  1840. 

5.  Discovery  of  the  bacterial  origin  of  certain  diseases — 1862  to  1880. 

6.  Introduction  of  small-pox  vaccination  into  England  by  Jenner  in  1796. 

7.  Development  of  antiseptic  surgery  or  Listerism — 1875. 

8.  Period  of  great  activity  in  pathological  bacteriology — 1880  to  1890. 

9.  Discovery  of  the  causes  of  immunity  to  disease,  antitoxin  of  diphtheria 
and  other  antitoxins,  serum  therapy,  etc. — 1886  to  1894. 

10.  Introduction  of  the  use  of  certain  bacteria  in  commerce  and  agri- 
culture. 

11.  Discovery  of  opsonins  and  the  use  of  bacterial  vaccines.     Reintro- 
duction  of  Koch's  lymph  in  the  treatment  of  tuberculosis. 

Useful  Works  of  Reference  to  Bacteriology  and  Related  Topics. 

The  following  references  are  selected  for  collateral  reading.  A  few  of 
these  works  are  rare,  and  can  be  found  only  in  some  of  the  leading  libraries. 
A  reading  of  these  and  other  related  works  will  serve  as  a  supplement  to  this 
text-book.  It  is  not  intended  to  imply  that  all  of  the  works  cited  should  be 
procured.  Others  besides  those  mentioned  may  be  consulted  as  oppor- 
tunity presents  itself.  Some  of  them  can  be  obtained  from  public  libraries; 
others  may  be  ordered  through  the  local  book  dealer,  and  a  few  may  be 
borrowed  from  professional  friends. 

HENRY  BAKER.     The  Microscope  Made  Easy.     London.     1743. 

Like  the  work  of  R.  Hoke,  this  is  of  great  historical  interest,  and  is  quite  rare. 
Much  of  it  is  a  copy  of  the  work  of  Leeuwenhoek. 

B.   M.   BOLTON  (H.   U.   Williams).     A  Manual  of  Bacteriology.     P.  Blakiston's  Son 
&  Co.,  Philadelphia.  1910. 

A  most  excellent  work  for  medical  students,  also  of  value  to  students  of  pharmacy. 
H.  W.  CONN.  Agricultural  Bacteriology. 

This  is  a  most  excellent  little  work  treating  of  bacteria  in  water,  in  the  soil,  in  farm 
products,  in  the  dairying  industry,  and  in  plants  and  domestic  animals.     It  is  well  written 
in  a  simple,  clear  style. 
H.  W.  CONN.     Bacteria,  Yeasts  and  Moulds  in  the  Home.     Ginn  &  Co.     1903. 

This  is  <of  special  value  to  the  pharmacist,  as  the  organisms  described  may  also  be 
found  in  pharmaceutical  preparations. 
H.  W.  CONN.     The  story  of  Germ  Life.     D.  Appleton  &  Co.,  New  York.     1905. 

Very  useful  and  interesting  general  reading  on  bacteriology. 
S.  M.  COPEMAN.     Vaccination,  Its  Natural  History  and  Pathology.     London.     1899. 

Of  historical  interest,  besides  explaining  the  subject  very  fully. 
E.  M.  CROOKSHANK.     Text-book  of  Bacteriology.     Philadelphia.     1897. 

This  is  much  used  as  a  college  text-book  on  bacteriological  technic.  Not  especially 
adapted  for  general  reading.  Would  serve  as  a  laboratory  guide. 


HISTORICAL.  19 

CHAS.  S.  DOLLEY.     The  Technology  of  Bacteria    Investigation.     S.  E.  Casino  &  Co., 
Boston.     1885. 

Good  reference  work  on  bacteriological  technic.     Somewhat  out  of  date. 
PAUL  EHRLICH   (Chas.   Bolduin).     Collected   Studies  on  Immunity.     John  Wiley  and 
Sons,  New  York.     1906. 

An  extensive  discussion  of  the  theories  pertaining  to  the  action  of  toxins  and  anti- 
toxins.    Ehrlich's  side-chain  theory  is  quite  fully  treated.     The   subject  is  too  technical 
for  the  average  reader,  and  is  of  great  value  only  to  the  specialist  in  this  branch  of  bacteri- 
ology. 
DAVID  ELLIS.     Outlines  of  Bacteriology.     London,  New  York  and  Calcutta.     1909. 

An  excellent  English  work  on  general  bacteriology  especially  valuable  from  the  tech- 
nical and  agricultural  standpoints. 

J.  W.    EYRE.     The    Elements   of   Bacteriological   Technic.     W.  B.    Saunders  &  Co., 
Philadelphia.     1902. 

An  excellent  laboratory  guide  for  the  use  of  medical,  dental,  and  technical  students, 
and  which  will  serve  many  purposes  of  the  student  of  pharmacy. 
DANIAL  DE  FOE.     History  of  the  Plague  in  London.     London.     1857. 

Of  historical  interest.     Well  written. 

W.  D.  FROST.     A  Laboratory   Guide  in   Elementary    Bacteriology.      Macmillan   Com- 
pany, New  York.     1903. 

An  excellent  laboratory  guide.     It  contains  no  general  information  regarding  bacteria, 
and  can  be  used  profitably  only  under  the  guidance  of  a  laboratory  instructor. 
\Y.  II.  HARROCKS.     An  Introduction  to  the  Bacteriological  Examination  of  WTater.     J.  A. 

Churchill,  London.     1901. 

Of  value   to   anyone    interested   in   the   bacterial   contamination  of   water  supplies; 
also  useful  for  general  reading. 
ROBERT  HOOKE.     Micrographia.     London.     1665. 

A  very  rare  and  very  interesting  work  treating  of  the  earliest  discoveries  through  the 
use  of  the  microscope.  Some  of  the  illustrations  are  excellent.  Of  great  historical  value 
and  interest.  Can  be  found  only  in  a  few  of  the  larger  university  and  public  libraries. 
In  P^nglish. 

L.  O.  HOWARD.     Mosquitoes:  How  They  Live  and  How  They  Carry  Disease.     McClure, 
Phillips  &  Co.,  New  York.     1901. 

Contains  valuable  information  regarding  these  pests  and  how  they  carry  diseases. 
Of  special  value  in  yellow  fever  and  malarial  districts. 

E.  O.  JORDAX.     A    Text-book  of    General    Bacteriology.     W.     B.     Saunders    &    Co., 

Philadelphia.   1908. 
For  medical  students.     Contains  much  information  of  interest. to  the  pharmacist. 

F.  LAFAR  (Salter).     Technical  Mycology.     London.     1903. 

Rather  technical  for  general  reading.     Treats    of    fermentation  and    fermentation 
products,  use  of  yeast  organisms  and   bacteria  in   the  industries,   etc.     Especially  valu- 
able to  those  interested  in  beer-making,  etc.,  the  dairying  industry,  etc. 
MILLARD  LANGFELD.     Infectious  and  Parasitic    Diseases,  Including   Their  Cause  and 
Manner  of  Transmission.     P.  Blakiston's  Son  &  Co.,  Philadelphia.     1907. 

Contains  much  valuable  information  on  preventive  medicine,  sources  of  infection, 
disinfectants   and   disinfection,    animal   parasites,  etc.     Excellent   collateral  reading  for 
the  pharmacist. 
AM <>\  VAN  LEEUWENHOEK.     Arcana  Naturae.     Four  volumes.     London.     1656. 

This  is  by  far  the  most  important  historical  work  on  the  use  of  the  microscope.     In 
Latin.     Some  very  good  illustrations.     Very  rare;  found  in  a  few  libraries  only. 
K.  C.  MKX.     Alikroskopische  Wasser  Analyse.     Berlin.     1898. 


20  PHARMACEUTICAL    BACTERIOLOGY. 

An  excellent  German  work  treating  of  the  bacteriological  investigation  of  drinking 
water  and  sewage  waters. 
GEO.  NEWMAN.     Bacteria.     G.  Putnam's  Sons,  New  York.     1899. 

Treats  of  bacteria  in  industrial  processes,  bacteria  in  public  health,  in  nature,  in  soil, 
etc.     A  very  valuable  work,  excellent  for  general  reading. 
T.  M.  PRUDDEN.     The  Story  of  Bacteria.     G.  P.  Putnam's  Sons,  New  York.     1889. 

Very  interesting  reading  on  general  bacteriology  and  on  the  relationship  of  bacteria 
to  health  and  disease. 

M.  J.  ROSENAU.     The  Bacteriological  Impurities  of  Vaccine  Virus.     U.  S.  Public  Health 
and  Marine  Hospital  Service.     Hygienic  Lab.  Bui.,  No.  12.     1903. 

Of  special  interest  to  pharmacists.  It  should  be  borne  in  mind,  however,  that  since 
the  publication  of  this  report  the  methods  of  vaccine  manufacture  have  been  modified 
somewhat,  and  the  figures  and  results  given  may  no  longer  apply. 

M.  J.  ROSENAU.     An  Investigation  of  a  Pathogenic  Microbe  of  Rats  and  Mice  (B.  typhi- 
murium  Danysz.).     Washington,  D.  C.     1903. 

This  treatise  is  also  of  special  interest  to  pharmacists,  as  the  microbe  referred  to  is  the 
active  ingredient  of  several  patented  rat  and  mouse  exterminators  sold  under  proprietary 
names  as  Azoa  (Parke,  Davis  &  Co.),  Rattite,  Mouratus  (Pasteur  Co.),  etc.  These 
exterminators  are  still  under  investigation,  testing,  etc.,  and  the  findings  in  the  above 
report  should  not  be  considered  final  or  conclusive. 

W.   G.    SAVAGE.     The   Bacteriological  Examination   of   Water   Supplies.     Philadelphia. 
1906. 

A  valuable  treatise.     Contains  a  citation  of  the  more  valuable  literature  on   the 
subject.     An  excellent  laboratory  guide  for  the  specialist. 
DR.  C.  STICH.     Bacteriologie  und  Sterilization  im  Apothekerbetrieb.     Berlin.     1904. 

In  German  only.  Contains  many  valuable  suggestions  but  too  incomplete  and  too 
much  lacking  in  detail  for  the  student. 

E.  R.  STITT.     Practical  Bacteriology,  Blood  Work  and  Animal  Parasitology.     P.  Blakis- 
ton's  Son  &  Co.,  Philadelphia.     1909. 

Primarily  for  medical  students,  especially  those  interested  in  the  parasitology  of  the 
tropics.     Complete  on  methods.     Full  details  regarding  blood  work  and  use  of  hemacy- 
tometer. 
JOHN  TYNDALL.     Floating  Matter  in  the  Air.     London.     1881. 

A  very  interesting  popular  work  on  the  micro-organisms  of  the  air  and  their  relationship 
to  fermentation  and  putrefaction.  For  general  information. 

NOAH  WEBSTER.     A  Brief  History  of  Epidemics  and  Pestilential  Diseases.     Two  volumes. 
Hartford.     1799. 

Of  great  historical  ^interest,  though  entirely  antiquated  and  of  no  scientific  value. 


CHAPTER  III. 
GENERAL  MORPHOLOGY  AND  PHYSIOLOGY  OF  BACTERIA. 

Bacterium  (plural,  bacteria)  is  a  misleading  term,  though  firmly  estab- 
lished in  general  usage.  It  means  "a  small  rod,"  the  name  being  applied 
because  it  was  believed  that  these  minute  organisms  were  mostly,  if  not  all, 
rod-shaped.  This  is  not  the  case,  as  will  be  explained  later.  Further- 
more, the  term  is  used  in  a  generic  sense,  and  again  applied  to  the  group  of 
organisms  as  a  whole.  This  causes  confusion.  Therefore,  the  generic  term 
Bacterium  is  now  abandoned  and  the  term  Bacillus  is  used  to  include  all  of  the 
micro-organisms  which  are  rod-shaped  although  generic  sub-divisions  are 
being  made  of  this  now  very  large  group.  The  term  "microbes"  (micros, 
small,  and  bios,  life)  or  micro-organisms  would  be  far  more  suitable  than  the 
term  "  bacteria,"  as  applied  to  the  entire  group  of  organisms  included  in  the 
subject  of  bacteriology.  Microbiology  is  no  doubt  more  correctly  descriptive 
than  bacteriology,  but  the  latter  term  is  so  firmly  established  in  general  usage 
that  it  would  be  unwise  to  urge  a  change  at  the  present  time. 

Whereas  the  general  morphology  of  microbes  is  apparently  quite  simple, 
the  physiology  and  chemistry  is  extremely  complex,  and  as  yet  not  fully 
understood.  The  morphological  simplicity  is  no  doubt  only  apparent,  and 
not  real.  Perhaps,  with  the  greater  perfection  of  the  compound  microscope, 
we  may  discover  marked  structural  differences  which  thus  far  have  escaped 
our  notice. 

i.  Classification  Of  Microbes. 

Microbes  are  the  smallest  of  the  known  living  organisms.  It  is  wholly 
impossible  to  see  the  single  individual,  even  the  largest,  with  the  naked  eye. 
The  rod-shaped  microbes  (bacilli)  range  from  0.5^  to  10/1  in  length.  Some 
are  so  minute  as  to  pass  through  the  pores  of  the  finest  elay  filters  (microbes 
of  foot  and  mouth  disease).  To  study  them  a  good  compound  micro- 
scope is  absolutely  necessary,  though,  as  stated  in  the  historical  review 
(Period  II),  Leeuwenhoek  and  others  observed  the  larger  forms  under  the 
simple  microscope. 

The  systematic  position  of  microbes  has  from  time  to  time  received  much 
attention.  The  great  majority  of  biologists  now  unhesitatingly  class  them 
as  plants,  belonging  to  the  group  fungi.  It  cannot  be  denied,  however,  that 
their  origin  (phylogeny)  is  still  shrouded  in  mystery.  Some  suggest  that  they 
are  derived  from  degenerate  algal  forms,  in  common  with  most  of  the  fungi, 
while  others  declare  that  they  in  all  probability  originated  as  microbes.  A 

21 


22  PHARMACEUTICAL    BACTERIOLOGY. 

few  of  the  philosophical  biologists,  as  Ernst  Haeckel,  place  them  in  a  separate 
group,  the  Monera,  which  is  supposed  to  form  the  connecting  link  between 
plants  and  animals. 

Without  entering  into  lengthy  discussion,  we  shall,  in  conformity  with  the 
opinion  of  the  majority,  class  them  as  plants,  belonging  to  the  lowest  of  the 
group  fungi  (the  fungi  includes  rust,  smuts,  cup  fungi,  moulds,  spot  fungi, 
toad-stools,  etc.),  namely,  the  Schizomycetes  or  fission  fungi,  so  called  because 
they  multiply  by  fission  or  division.  They  are  related  to  the  yeasts,  though 
somewhat  lower  in  the  scale  of  evolution.  They  are  single-celled,  each  cell 
forming  a  complete  living  unit,  though  the  several  units  may  be  variously 
arranged  into  chains  or  clusters  or  groups  known  as  zoogolea. 

The  scientific  grouping  of  microbes  is  as  yet  very  unsatisfactory  because 
so  little  is  known  of  their  ultimate  morphology,  their  physiology  and  chemis- 
try. Some  have  attempted  to  classify  them  as  to  form,  others  as  to  occur- 
rence, as  to  action,  etc.  Thus,  we  have: 

a.  Micrococci  or  Coccaceae. — Globular  or  non-elongated  microbes. 

b.  Bacilli  or  Bacteriaceae. — Cells  more  or  less  elongated.     Rod-shaped 
microbes. 

c.  Spirillae  or  Spirillaceae. — Cells  elongated  and  more  or  less  spirally 
twisted.     Or,  we  may  have: 

a.  Bacteria  of  earth. 

b.  Bacteria  of  air. 

c.  Bacteria  of  water. 
Or,  again: 

a.  Chromogenic. 

b.  Zymogenic. 

c.  Pathogenic,  etc. 

These  artificial  groupings  could  be  extended  indefinitely,  but  such  sys- 
tems of  classification  would  be  as  unsatisfactory  as  they  are  unscientific.  The 
best  system  makes  use  of  all  of  the  known  facts  of  bacteriology.  Several  such 
systems  have  been  proposed  from  time  to  time,  but  the  new  discoveries  along 
bacteriological  lines  make  it  necessary  to  change  them  in  the  course  of  two  or 
three  years.  Migula,  Fischer,  Eisenberg  and  others  have  proposed  general 
systems,  and  a  host  of  investigators  have  submitted  more  limited  group  sys- 
tems. The  following  classification  will  serve  to  convey  some  idea  as  to  the 
structural  characteristics  of  the  more  important  groups: 

BACTERIA    OR   MICROBES. 

(Schizomycetes  or  Fission  Fungi.) 

I.  Family  COCCACEAE. — Micrococci.  Cells  globular  or  not  elongated. 
Division  in  two  or  three  directions  of  space.  Spore  formation  rare. 


GENERAL    MORPHOLOGY  AND    PHYSIOLOGY    OF    BACTERIA.  23 

1 .  Micrococcus. — Cells   spherical   or  biscuit-shaped.     Division    in    one 
direction  of  space.     With  or  without  flagellae.     A  large  genus,  represented 
by  numerous  species,  pathogenic  and  non-pathogenic,  chromogenic,  zymo- 
genic,  etc. 

2.  Streptococcus. — Generic  limitation  not  clearly  denned.     Often  merely 
chain  forms  of  above,  resulting  from  cohesion  of  cells  dividing  in  one  direction 
of  space. 

3.  Sarcina. — Division  in  three  directions  of  space.     Cells  often  in  fours 
(Tetracoccus) — as  for  example,  the  sarcina  of  the  stomach.    With  or  without 
flagellae. 

II.  Family    BACTERIACE.E. — Bacilli.     Cells    more    or    less    elongated, 
cylindrical,  straight ;  some  are  somewhat  curved  or  irregular  in  outline.     With 
or  without  flagellae.     Endospore  formation.     Transverse  septation. 

1.  Bacillus. — Variable  in  size  and  length  of  cell.     Numerous  flagellae. 
Endospore  formation  common.     A  very  large  group,  to  which  belong  many 
of  the  most  important  microbes.     Includes  the  old  genus  Bacterium. 

2.  Pseudomonas. — Said  to  have  only  polar  flagellae.     Doubtful  genus,  by 
many  relegated  to  the  group  bacillus. 

III.  Family    SPIRILLACE.E. — Spirillae.     Cells    elongated     and    spirally 
twisted.     Transverse  septation.     Body  fixed,  with  polar  flagellae. 

1.  Spirillum. — Numerous  polar  flagellae.     Large  group. 

2.  Microspira. — Few  polar  flagellae.     A  group  Spirosoma  is  said  to  be 
without  flagellae. 

IV.  Family  SPIROCHETACE^:. — Spirocheta.     Long,    single-celled,   flex- 
ible,   spirally   twisted    threads    without  flagellae.     One    genus — Spirocheta. 
(Some  authorities  place  these  organisms  in  the  animal  kingdom  with   the 
Protozoa.) 

V.  Family  MYCOBACTERIACE.E. — Filamentous  organisms,  perhaps  form- 
ing a  connecting  link  between  bacteria  proper  and  the  lower  filamentous 
fungi.     Cells  filamentous  but  not  enclosed  in  a  sheath.     To  this  family 
belong  the  groups  Mycobacterium  and  Actinomyces  (ray  fungus).     No  flag- 
ellae  have  been  observed.     Mostly  transverse  septation.     Gonidial  (spore) 
formation  has  been  observed. 

VI.  Family   CHLAMYDOBACTERIACE^E. — Resembling  above  family,   but 
the  cell  filaments  are  enclosed  in  a  sheath.     The  following  not  very  clearly 
defined  groups  are  recognized:     Cladothrix,  Crenothrix,  Phragmidiothrix, 
and  Thiothrix. 

VII.  Family  BEGGIATOACE.E. — Beggiatoa.     Family  characters  not  clearly 
defined.     Motile,  though  no   flagellae  have  been  observed.     Beggiatoa  is 
the  most  important  genus. 

The  uncertainty  in  the  systematic  grouping  of  microbes  need  not  cause 
worry,  as  even  the  leading  specialists  do  not  give  the  matter  any  con- 


24  PHARMACEUTICAL   BACTERIOLOGY. 

siderable  attention,  for  the  simple  reason  that  their  time  is  taken  up  by  matters 
of  far  greater  importance,  namely,  the  determination  of  the  role  which  the 
microbe  plays  in  the  life  economy.  Should  the  student  ever  be  placed  in 
position  to  justify  him  in  attempting  to  identify  a  given  microbe,  he  will 
find  an  extensive  literature  which  will  aid  him  in  his  efforts.  Undoubtedly 
in  time  there  will  be  a  fairly  simple,  scientific,  and  complete  system  of  classi- 
fication of  all  known  bacteria.  As  yet  such  a  system  does  not  exist. 

2.  General  Morphology  of  Microbes. 

As  already  stated,  the  morphology  of  microbes  is  simple.     They  consist 
of  a  single  cell  composed  of  cell-wall  and  cell-contents.     The  cell-wall  con- 


FIG.  4. — Illustrating  the  general  morphology  of  microbes,  a,  showing  general 
structure  of  a  bacillus,  endospore  formation,  and  development  of  new  bacillus  from  a 
spore;  b,  showing  manner  of  transverse  septation;  c,  arrangement  of  flagellae,  single  unipolar, 
single  bipolar;  and  multiple,  polar  and  general;  d,  cocci;  e,  flagellae  of  cocci;  / 
spirillum  with  single  polar  cilia. 


sists  of  cellulose,  and  is  very  thin;  stains  readily  with  the  various  bacterial 
stains.  The  chief  cell-contents  is  the  cytoplasmic  or  protoplasmic  living 
base  commonly  designated  as  the  nucleoplasm,  which  is  of  a  granular  nature, 


GENERAL   MORPHOLOGY  AND   PHYSIOLOGY   OF   BACTERIA. 


FIG.  5. — Illustrating  the  general  morphology  of  Coccaceae.  a,  b,  micrococci  (a)  differing 
in  size,  showing  chain  formation  or  streptococci  (b);c,  diplococcus;  d,  diplococcus;e,  tetra- 
coccus;/,  gelatinized  tetracoccus;  g,  gelatinized  diplococcus. 


FIG.  6. — General  morphology  of  Bacteriaceae.  a,  b,  c,  d,  bacilli  differing  in  size  and 
orm;  c,  shows  curved  bacilli  like  those  of  Asiatic  cholera;  e,  hay  bacillus  (B.  subtilis); 
/,  Y-shaped  or  branched  bacilli,  as  of  clover  root  nodules;  g,  drum-stick  (Trommelsch lager) 
bacilli,  as  of  tetanus — form  due  to  the  enlarged  endospores. 


26 


PHARMACEUTICAL    BACTERIOLOGY. 


and  by  some  is  supposed  to  be  a  nucleus  in  a  divided  state.  A  nucleus 
proper  does  not  exist,  or,  rather,  has  not  been  demonstrated.  The  cyto- 
plasm, as  a  rule,  stains  quite  readily..  Distributed  through  the  cytoplasm 
may  be  found  various  substances,  elaborated  by  cytoplasmic  activity. 
Polar  granules  (metachromes  or  Babes-Ernest  granules)  have  been  observed. 
Sulphur,  fat,  pigment,  chlorophyll,  etc.,  may  be  found. 

The  cell-walls  of  many  species  undergo  a  gelatinous  change.  This 
change  may  affect  the  outer  layers  only,  or  it  may  involve  the  entire  thickness 
of  the  wall,  forming  the  gelatinous  substances  noticeable  in  bacterial  cultures 
and  in  other  substances  (stringy  cultures,  stringy  milk,  etc.) .  This  gelatin- 


FIG.  7.— General  morphology  of  the  Spirillaceae.  a,  S-shaped  or  single  spiral;  b, 
double  spiral;  c,  multiple  spirals;  d,  slender  threads;  a  and  b  have  fixed  bodies,  motion 
being  caused  by  flagellae;  c  and  d,  bodies  flexible,  motion  not  due  to  flagellae. 

ous  substance  also  causes  the  individual  organisms  to  cling  to  each  other, 
thus  causing  the  formation  of  the  peculiar  zooglea  masses  in  natural  as  well 
as  in  artificial  culture  media. 

The  cilia  or  flagellae  are  very  delicate  threads,  supposed  to  extend  from 
the  cell-plasm,  through  the  cell-wall,  into  the  surrounding  medium.  The 
delicate  threads  are  probably  cytoplasmic  in  nature,  and  by  their  rapid 
vibratory  motion  enable  the  microbe  to  move  about  within  liquid  media. 
Some  microbes  are  apparently  without  flagellae,  nor  is  it  definitely  deter- 
mined that  all  motile  microbes  have  flagellae.  Some  authorities  are  inclined 


GENERAL-  MORPHOLOGY  AND    PHYSIOLOGY   OF   BACTERIA.  2J 

to  the  belief  that  perhaps  nearly  all,  if  not  all,  micrococci  and  bacilli  have 
active  motion  under  certain  conditions.  This  makes  it  clear  that  the  attempt 
to  group  microbes  into  motile  and  non-motile  must  result  in  failure.  The 
attempt  to  make  generic  distinctions  based  upon  the  absence  or  presence  of 
few  or  many  flagellae,  upon  the  existence  of  polar  or  non-polar  flagellae,  etc., 
is  also  unsatisfactory.  Special  staining  methods  are  necessary  to  demon- 
strate the  presence  or  absence  of  flagellae.  Some  investigators  declare  that 
it  is  almost  impossible  to  demonstrate  them  ocularly.  That  they  do  exist  is 
fully  demonstrated,  but  it  is  not  demonstrated  to  any  degree  of  satisfaction 
that  it  is  practicable  to  make  finely  drawn  numerical  and  structural  distinc- 
tions in  the  flagellae  of  the  different  species  of  microbes. 

The  rate  of  motion  of  bacteria  has  been  measured.  The  cholera  bacillus 
moves  at  the  rate  of  18  cm.  per  hour.  The  typhoid  bacillus  is  slower  moving 
a  distance  of  4  mm.  in  one  hour.  The  rate  of  motion  in  one  and  the  same 


<  m 


Fig.  8. — Illustrating  polymorphism  or  pleomorphism.    Involution  forms  of  the  bacillus  of 
Asiatic  cholera.     (Williams.) 

species  is,  however,  variable,  being  comparatively  rapid  at  one  time  under 
certain  conditions  of  food  supply,  warmth,  etc.,  and  at  other  times  com- 
paratively slow. 

When  the  microbe  approaches  the  end  of  the  life  cycle,  or  when  the  con- 
ditions for  growth  and  septation  are  no  longer  good,  spore  formation  is  apt 
to  take  place.  This  spore  formation  is  of  two  kinds,  endospore  formation 
and  arthrospore  formation.  The  former  predominates,  and  occurs  largely 
in  the  group  bacilli,  though  it  is  also  noticeable  among  the  micrococci  and 
the  spirillae.  Endospores  are  usually  spherical,  though  they  may  be  slightly 
elongated,  and  usually  occur  near  one  end  of  the  cell,  and  usually  there  is 
only  one  in  each  cell.  Generally  the  diameter  of  the  spore  is  equal  to  or 
somewhat  less  than  the  diameter  of  the  cell-lumen.  Sometimes,  however, 
the  diameter  of  the  spore  exceeds  that  of  the  cell-lumen,  causing  a  charac- 
teristic bulging,  as  in  the  tetanus  bacillus  (drum-stick  bacillus,  Trommel- 
hldger  Bacillus).  The  spore  is  formed  from  the  cytoplasm,  and  differs 


28  PHARMACEUTICAL   BACTERIOLOGY. 

from  it  in  its  higher  refractive  index  and  its  peculiar  resistance  to  the  action  of 
stains.  As  soon  as  spore  formation  is  complete,  the  rest  of  the  cytoplasm 
dies,  the  cell-  wall  disintegrates,  and  the  spore  is  thus  set  free.  Spores  have 
a  remarkable  resisting  power  to  high  temperatures  and  other  unfavorable 
conditions.  In  a  dry  atmosphere  they  may  lie  dormant  for  a  long  time, 
even  several  years.  Boiling  from  one  to  two  hours  does  not  kill  some  of 
them  (spores  of  hay  bacillus)  .  As  soon  as  the  spores  are  placed  in  suitable 
media  (adequate  warmth,  moisture,  and  food  supply)  they  develop  into  new 
individuals,  which  continue  to  septate  until  spore  formation  again  takes 
place. 

^Vt  ff 


i 


FIG.  9.  —  Illustrating  polymorphism  or  pleomorphism.  a  to  d,  inclusive,  represent 
different  forms  of  the  same  organism  —  the  Diphtheria  bacillus.  (See  also  Figs.  46-50 
inclusive.) 

Arthrospore  formation  is  less  common,  and  occurs  mostly  among  the 
micrococci.  The  entire  cell  is  converted  into  a  spore,  which  becomes  some- 
what enlarged  and  encapsuled,  in  which  state  it  is  enabled  to  tide  over  cer- 
tain conditions  unfavorable  to  normal  growth  and  septation.  Arthrospore 
formation  is  not  well  understood  as  yet.  It  may  also  be  that  some  of  the 
phenomena  described  as  arthrospore  formations  are  in  reality  endospore 
formations. 

The  classification  given  above,  into  families  and  genera,  and  Figs.  2  to  10, 
inclusive,  will  serve  to  give  a  fairly  good  idea  of  the  general  structural  char- 
acteristics of  microbes. 

3.  General  Physiology  of  Microbes. 

Microbes,  in  common  with  living  things  generally,  spring  from  pre- 
existing parents,  take  in  and  assimilate  food,  grow  and  multiply,  and  finally 
die.  The  rate  of  growth  and  of  multiplication  (septation  or  division)  varies 


GENERAL   MORPHOLOGY  AND   PHYSIOLOGY   OF   BACTERIA. 


29 


somewhat,  depending  on  temperature,  moisture,  and  food  supply.  The 
average  life  of  one  individual  (from  septation  to  septation)  is  perhaps  thirty 
minutes.  Under  favorable  condition  the  period  is  much  shortened.  This  life 
period  of  the  individual  cell  must  not  be  confounded  with  the  life  cycle  of  the 
individuals  resulting  from  a  single  cell  or  parent.  It  is  known  that  under 
uniform  conditions  of  temperature,  moisture,  food  supply,  and  the  environ- 
ment generally,  the  progenations  from  a  single  parent  cell  show  an  increasing 
rate  of  septation,  a  stationary  period,  followed  by  a  gradual  decline,  ending 
in  total  cessation  of  all  septation,  and  in  death.  These  life  cycles  have  not 


CO 


a 


'%. 


FIG.  10. — Illustrating  zooglea  formation,  a,  bacillar  aggregates  resulting  from 
cohesion;  b,  aggregates  resulting  from  cohesion  of  bacilli  with  gelatinized  cell- walls;  cy 
streptococcus  formation  resulting  from  the  septation  of  a  coccus  form;  d,  cohering  cocci 
forms;  e,  bacilli  united  end  to  end  (resulting  from  septation),  enclosed  in  a  gelatinous  coat; 
/,  bacillar  thread  enclosed  in  gelatin;  g,  mycobacterial  form;  h,  irregular  cell  forms,  as 
My  coder  ma  aceti. 

yet  been  carefully  determined;  in  fact,  they  are  but  little  understood.  It  is 
highly  probable  that  the  cycles  of  existence  play  a  very  important  part  in  the 
course  and  development  of  diseases  of  bacterial  origin. 

Whereas  the  period  from  one  septation  to  another  septation  is  very  short, 
the  life  cycle  referred  to  is  often  quite  long,  perhaps  months  and,  under 
certain  conditions,  lasting  for  years.  The  period  of  the  life  cycle  can  be 
modified  artificially  by  food  supply,  chemicals,  etc. 


30  PHARMACEUTICAL    BACTERIOLOGY. 

Investigators  have  succeeded  in  prolonging  the  life  cycle  of  Paramedum. 
Normally  P.  caudatum  dies  out  in  about  175  generations;  but  by  applying 
alcohol  (1-5000  to  1-10,000)  the  cycle  has  been  increased  to  860  generations. 
Very  dilute  solutions  of  strychnine  gave  similar  results.  If  the  life  cycle  or 
vital  impulse  of  these  simple  organisms  can  be  prolonged  it  is  probable  that 
similar  effects  can  be  produced  in  higher  organisms.  Numerous  investi- 
gators have  from  time  to  time  sought  after  agents  which  might  inhibit  the 
senile  changes  in  cells  and  circulatory  system  (arteriosclerosis)  but  thus 
far  without  conclusive  results.  It  is,  however,  highly  probable  that  within  a 
comparatively  short  time  means  may  be  found  to  prolong  the  life  of  the 
higher  animals  from  10  to  20  per  cent,  and  even  more. 

Microbes  feed  upon  organic  substances  generally.  Those  which  feed 
upon  dead  organic  substances  are  said  to  be  saprophytic;  those  feeding  upon 
living  substances  are  said  to  be  parasitic.  If  they  can  live  on  dead  organic 
substances  only,  they  are  obligatively  saprophytic;  if  they  can  feed  on  both 
dead  and  living  organic  substances,  they  are  facultatively  saprophytic,  or, 
vice  versa,  facultatively  parasitic.  The  great  majority  of  microbic  parasites 
are  facultatively  so,  as  is  evidenced  by  the  fact  that  they  can  be  grown  in 
artificial  culture  media.  Many  of  the  microbic  saprophytes  will  develop  on 
living  substances  under  certain  conditions,  thus  showing  that  they  are  facul- 
tatively parasitic.  It  is  no  doubt  true  that  no.  known  microbic  parasite 
actually  feeds  upon  the  living  substances  of  the  various  hosts,  since  the  cyto- 
plasm is  in  all  instances  dead  before  it  is  taken  up  and  assimilated  by  the 
microbe.  It  would  therefore  be  more  correct  to  say  that  parasitic  microbes 
are  biologically  associated  with  living  organisms,  while  the  saprophytes  are 
biologically  associated  with  dead  organic  substances,  and  that  they  all 
feed  upon  and  assimilate  dead  organic  substances.  In  certain  mutualistic 
symbioses  (as  in  the  root  nodules  of  the  Leguminosse)  the  biological  rela- 
tionship of  microbe  and  host  plant  is  very  intimate,  but  there  is  no  actual 
interchange  of  living  material. 

All  microbes  require  moisture  and  warmth  (comparatively  speaking) 
for  their  development,  although  they  are  enabled  to  withstand  greater  ex- 
tremes of  heat  and  cold  than  other  organisms.  The  temperature  of  liquid 
air  (about  — 270°  F.)  does  not  kill  them  at  once,  and  the  spores  may  be  boiled 
for  some  time  without  destroying  their  germinating  power.  Cold  (freezing 
temperature)  promptly  checks  growth  and  septation,  and  so  does  dryness  and 
excessive  warmth,  although  life  may  not  be  destroyed.  The  majority  of 
microbes  develop  most  actively  at  a  temperature  of  25°  C.,  a  few  species 
develop  more  actively  at  a  lower  temperature  (20°  C.),  and  a  few  others  at  a 
higher  temperature  (38°  C.).  Those  which  develop  at  a  temperature  rang- 
ing from  o°  C.  to  30°  C.  are  said  to  be  cold  loving  (psychrophile),  from  10°  to 
45°  C.,  mesophile,  from  40°  to  70°  C.,  thermophile.  Thermophile  species  are 


GENERAL   MORPHOLOGY  AND    PHYSIOLOGY   OF   BACTERIA.  31 

found  in  decaying  vegetable  matters,  whereas  psychrophile  species  are  found 
in  cold  water  and  cold  soils. 

Bacterial  life  processes  result  in  the  formation  of  many  substances,  some 
of  which  are  of  the  greatest  importance.  It  is  impossible  to  estimate  prop- 
erly the  enormous  tasks  performed  by  these  minute  organisms,  nor  shall  we 
at  this  time  make  any  attempt  to  set  forth  the  great  good  and  the  apparent 
great  harm  done  by  them.  We  need  only  state  that  without  rotting  microbes 
soil  formation  would  be  impossible,  and  without  soil,  higher  plant  and  animal 
life,  as  we  now  know  them,  would  be  impossible.  Without  plant  food 
digesting  microbes  crop  growing  would  be  impossible.  The  saltpeter  depos- 
its in  South  America  and  the  iron  deposits  of  the  Mesabi  range  of  Minnesota 
are  said  to  be  the  result  of  bacterial  action.  We  make  extensive  practical 
use  of  microbes  in  medical  practice,  in  the  dairying  industry,  etc. 

We  will  mention  ohly  a  few  substances  of  undoubted  microbic  origin. 
Ptomaines  and  toxalbumins  are  well-known  poisons  elaborated  by  sapro- 
phytic  microbes  which  feed  on  meats  and  other  organic  substances,  causing 
the  familiar  putrefactive  changes.  Pathogenic  microbes  elaborate  toxins 
to  which  are  due  the  manifestations  of  the  disease.  Acetic  acid,  lactic  acid, 
and  butyric  acid  are  elaborated  by  Bacillus  aceticus,  B.  acidi  lactici,  and 
B.  butyricus,  respectively.  Some  species  liberate  odoriferous  substances, 
others  gases,  coloring  substances,  phosphorescence,  etc.  The  phosphor- 
escence observed  on  the  ocean  is  supposed  to  be  due  to  bacteria  (Bacillus 
phosphorescent  indicus).  Phosphorescent  bacteria  occur  in  dead  fish  and 
in  meat.  Old  cultures  in  animal  nutrient  media  and  in  the  presence  of 
sodium  salts  are  phosphorescent  in  the  dark,  sufficiently  so,  to  have  sug- 
gested making  bacterial  lamps  and  signal  lights: 

It  has  been  suggested  that  certain  diseases,  of  which  the  causes  are  at 
present  unknown  (as  yellow  fever,  measles,  whooping  cough),  may  be  due 
to  organisms  so  small  as  to  be  invisible  (ultra  micro-organisms).  It  is  known 
that  the  virus  of  yellow  fever  will  pass  through  the  most  compact  clay  or  por- 
celain filter.  Attempts  have  been  made  to  demonstrate  the  presence  of 
ultra  micro-organisms  by  special  photomicrographic  methods,  aided  by 
special  illuminating  devices  (the  ultra  microscope  of  Siedentopf  and 
Szigmondy)  but  without  success.  Furthermore,  no  one  has  succeeded  in 
culturing  such  theoretically  surmised  organisms  in  artificial  media,  which 
would  certainly  render  them  visible  en  masse.  It  may,  however,  be  possible 
that  some  ultra-organisms  are  obligative  parasites  hence  will  not  develop  in 
artificial  media. 

The  biological  (symbiotic)  relationship  of  different  species  of  bacteria  to 
each  other  and  to  their  host  are,  in  many  instances  at  least,  not  well  under- 
stood. For  example,  it  is  not  clear  what  biological  relationship  the  different 
species  of  bacteria  in  a  mixed  infection  bear  to  each  other.  In  the  case  of  the 


32  PHARMACEUTICAL    BACTERIOLOGY. 

root  nodule  organisms  of  the  Leguminosae  it  is  known  that  there  is  a  mutu- 
ally beneficial  (mutualistic  symbiosis,  mutualism)  relationship  between  mi- 
crobe and  host  but  it  is  not  obligatively  so,  since  the  symbionts  can  exist 
independently  of  each  other.  In  most  diseases  due  to  microbic  invasion  there 
is  one  species  of  bacterium  which  acts  as  the  primary  cause.  It  is  known  that 
tuberculosis,  especially  the  pneumonic  form,  usually  shows  a  mixed  infec- 
tion, and  it  is  probable  that  the  associated  organisms  as  bacteria  and  higher 
fungi  act  as  predisposing  causes,  preparing  the  tissues  so  as  to  yield  more 
readily  to  the  invasion  of  the  primary  cause,  the  Bacillus  tuberculosis.  Such 
an  association  may  be  designated  compound  symbiosis,  in  which  the  relation- 
ship of  the  invading  organisms  (secondary  and  primary)  is  mutualistic  and 
the  relationship  of  these  to  the  host  is  antagonistic.  It  is  known  that  certain 
microbic  diseases  predispose  to  other  microbic  invasions,  thus  we  may  say 
that  these  organisms  are  mutualistically  disposed  toward  each  other. 

Since  it  is  possible  to  cultivate  most  disease  germs  in  and  upon  artificial 
culture  media  (hence  dead  organic  substances)  it  is  evident  that  they  are  only 
facultatively  parasitic. 

In  many  instances  the  biological  association  of  bacteria  and  higher  plants 
and  animals  is  loosely  mutualistic,  as  the  bacteria  upon  roots  and  rootlets  of 
all  plants  and  the  bacteria  lining  the  intestinal  tract  of  animals.  The  hay 
bacillus  (Bacillus  subtilis)  is  a  constant  associate  with  the  Graminege  and 
serves  an  important  function,  assimilating  or  binding  for  the  use  of  the  host 
plant,  the  free  nitrogen  of  the  air.  Certain  soil  organisms  (Bacillus  megatherium, 
B.  ellenbachiensis,B.mesentericus,B.  pyocyaneus,  B.  prodigiosus,  the  Azoto- 
bacter  group,  Clostridium  pastorianum,  certain  moulds  as  Aspergillus  niger 
and  Penicillium  glaucum)  are  capable  of  assimilating  the  free  nitrogen  of 
the  air  thus  enriching  the  soil  for  the  benefit  of  higher  plants. 


CHAPTER  IV. 

RANGE  AND  DISTRIBUTION  OF  MICROBES. 

Microbes  are  omnipresent  over  the  surface  of  the  earth.  In  number  and 
in  bulk  they  exceed  all  other  organisms  (plants  and  animals)  put  together. 
They  form  a  large  precentage  of  the  bulk  of  the  soil.  They  occur  in  the  air, 
in  water,  in  snow,  in  hail,  in  raindrops,  in  and  upon  plants,  in  and  upon 
animals.  All  substances  with  which  we  come  in  contact  are  likely  to  hold 
microbes.  Our  clothing  teems  with  them.  They  are  in  the  air  we  breath, 
in  the  food  we  eat,  and  in  the  liquids  we  drink.  The  floating  dust  particles 
of  the  air  carry  microbes;  the  particles  of  organic  matter  in  water  harbor 
microbes;  they  are  found  on  wood,  on  cloth,  on  paper,  on  metal,  glass,  and 
rock  surfaces,  in  fact  on  all  exposed  surfaces.  The  hands,  the  hair,  the 
entire  body  surface  of  man  and  of  the  lower  animals  contain  or  hold  mi- 
crobes. They  line  all  mucous  membranes.  The  mouth  cavity  is  a  veritable 
bacteriological  laboratory.  The  entire  intestinal  tract  teems  with  millions 
upon  millions  of  these  minute  beings. 

Each  animal  and  each  plant  has  a  microbic  flora  peculiar  to  itself.  Each 
portion  of  the  plant  or  animal,  again,  has  distinctive  bacterial  groups.  The 
microbic  flora  of  the  intestinal  tract  of  the  dog  is  different  from  that  of  the  pig, 
or  cat,  or  fowl,  or  man.  Certain  species  predominate  in  the  mouth  cavity, 
others  in  the  stomach,  still  others  in  the  small  intestine,  in  large  intestine,  etc. 

Microbes  are  found  on  the  highest  mountain  peaks  and  in  the  deepest 
valleys.  It  is,  however,  true  that  the  higher  atmospheric  strata  contain 
fewer  microbes  than  the  lower  strata.  The  deeper  layers  of  soil  contain 
fewer  microbes  than  the  upper.  The  atmosphere  of  the  country  contains 
fewer  microbes  than  that  of  the  cities  and  towns.  Since  sunlight  and 
absence  of  moisture  are  natural  enemies  of  microbes,  we  may  expect  to  find 
microbes  more  abundant  in  dark,  damp,  and  moist  places  and  areas.  Mi- 
crobes are  always  more  abundant  in  cellars,  basements,  dark  hall-ways,  and 
alleys  than  they  are  in  attics,  sunlit  living  rooms,  and  along  broad  boule- 
vards and  highways. 

Good  drinking  water,  whether  from  hydrant,  spring,  or  well,  contains  only 
a  comparatively  few  microbes,  from  fifty  to  one  hundred  per  c.c.,  or  even  less. 
Stagnant,  foul  water  teems  with  microbes,  besides  other  organisms,  such  as 
protozoa.  So-called  pure  milk  contains  comparatively  more  microbes  than 
pure  water.  The  average  good  milk  contains  as  many  as  30,000  microbes 

3  33 


34  PHARMACEUTICAL    BACTERIOLOGY. 

per  c.c.  Filthy  milk  may  contain  millions  of  microbes  per  c.c.  From  100,000 
to  3,000,000  microbes  per  c.c.  is  not  uncommon  in  some  milk  which  careless 
dairymen  declare  to  be  "good."  Soups,  broths,  etc.,  boiled  squash,  potatoes, 
meats,  and  cooked  organic  substances  generally,  if  allowed  to  stand  for  a  day 
or  two,  contain  many  living  microbes.  In  the  course  of  two  or  three  days, 
if  the  weather  is  warm,  these  substances  teem  with  microbes  and  are  rendered 
wholly  unfit  for  food  because  of  the  predominating  rotting  microbes  which 
develop  the  highly  poisonous  ptomaines. 

Microbes  do  not  live  and  multiply  in  aseptic  and  antiseptic  substances, 
such  as  strong  solutions  of  acids,  of  alkalies,  of  salts,  etc.  Used  and  dirty 
cups,  drinking  vessels,  milk  bottles,  dishes,  cooking  utensils,  knives,  spoons 
and  forks,  hold  numerous  microbes.  The  public  drinking  cup  has  been  the 
source  of  numerous  disease  infections.  Disease  is  carried  by  the  tools  of  the 
careless  dentist  and  by  the  clothing,  the  apparatus  and  the  clinical  thermom- 
eter of  the  indifferent  and  careless  physician.  The  hand-shaking  and  kiss- 
ing habits  spread  disease.  These  facts  are  generally  known  and  indicate  the 
wide  dissemination  of  the  different  kinds  of  microbes. 

From  the  foregoing  it  becomes  clear  that  microbes  are  present  almost 
everywhere,  and  that  it  is  impossible  to  escape  them.  It  is  the  aim  of  the 
science  of  bacteriology  to  distinguish  between  good  and  bad  microbes, 
between  those  which  are  desirable  and  those  which  are  undesirable,  between 
useful  and  harmful  microbes.  It  is  not  the  aim  of  the  science  of  bacteriology 
to  destroy  them  all,  or  to  devise  ways  and  means  to  escape  from  all  of  them. 
In  fact,  we  owe  our  very  existence  to  these  very  minute  organisms,  as  has 
already  been  explained. 

Under  certain  conditions  bacteria  multiply  very  rapidly.  Such  sub- 
stances as  meat,  milk,  and  organic  foods  of  all  kinds,  if  exposed  to  moisture, 
warmth  and  removed  from  sunlight,  soon  swarm  with  microbes.  Certain 
non-pathogenic  microbes,  as  the  root  nodule  bacteria  (of  the  Leguminosse) , 
multiply  very  rapidly  within  the  tissue  cells.  Others  multiply  upon  the 
exterior  of  roots  and  of  root  hairs,  where  they  no  doubt  serve  a  useful  pur- 
pose to  the  plant.  In  bacterial  diseases  of  plants  and  animals  the  microbes 
multiply  very  rapidly  and  form  large  aggregates,  as  a  rule.  To  pathological 
conditions  accompanied  by  extensive  and  general  bacterial  or  microbic  inva- 
sion, we  apply  the  term  bacteremia.  In  some  diseases  the  microbic  invasion 
remains  localized  and  yet  there  are  pronounced  general  or  systemic  effects, 
due  to  the  absorption,  into  the  system,  of  the  toxins  liberated  by  the  microbes. 
To  such  conditions  we  apply  the  term  toxemia.  Toxemia  may,  however,  also 
occur  in  bacteremia. 

Microbes  do  not  multiply  in  the  air  itself,  rather  upon  the  organic  dust 
particles  present,  provided  warmth  and  moisture  are  adequate. 

Since  microbes  multiply  rapidly,  perhaps  one  septation  in  from  twenty  to 


RANGE  AND   DISTRIBUTION    OF   MICROBES. 


35 


thirty  minutes,  it  is  evident  that  the  rate  of  numerical  increase,  under  favor- 
able conditions,  is  very  great.  Allowing  thirty  minutes  for  each  septation, 
there  would  be  a  colony  of  2,097,152  microbes  in  ten  hours,  developed  from 
a  single  cell,  or  about  75,000,000,000,000  cells  in  twenty-four  hours. 
However,  under  natural  conditions  septation  never  proceeds  in  such  uniform 
ratio.  All  manner  of  checks  to  septation  come  into  play  sooner  or  later 
which  may  finally  bring  about  complete  cessation  of  septation  and  sporulation. 


CHAPTER  V. 
BACTERIOLOGICAL  TECHNIC. 

As  may  readily  be  supposed,  the  minuteness  and  wide  distribution  of 
microbes  call  for  special  methods  of  study  and  examination.  Even  the 
largest  forms  are  far  below  the  ken  of  unaided  vision.  Their  general  dis- 
semination through  organic  substances  calls  for  special  methods  for  the 
separation  and  isolation  of  individuals  or  of  single  bacterial  cells.  The 
difficulties  of  technic  are  further  increased  by  the  resistance  of  spores  to 
various  agents  and  substances  which  are  readily  fatal  to  higher  organisms. 
The  methods  of  examination  are  also  greatly  complicated  by  the  marked 
polymorphism  of  many  species. 

Bacteriological  technic  comprises  the  use  of  glassware,  compound 
microscope,  and  other  apparatus,  a  thorough  knowledge  of  sterilization  and 
disinfection,  the  preparation  and  use  of  culture  media,  the  making  of  micro- 


FIG.  ii. — a,  Nest  of  beakers  and  reagent  bottles.  The  smaller  and  medium  size 
beakers  are  more  desirable  for  bacteriological  work.  The  reagent  bottles  are  for  Canada 
balsam,  stains,  clearing  fluid,  etc. 

bic  cultures,  and  the  study  of  cultures.  Methods  vary  greatly.  The  follow- 
ing represents  a  brief  summary  of  general  methods  which  are  noted  for 
simplicity  and  which  have  proven  very  satisfactory  after  years  of  testing. 


i.  Cleaning  the  Glassware. 

All  glassware,  such  as  test-tubes,  flasks,  beakers,  Petri  dishes,  pipettes, 
shells,  bottles,  etc.,  which  is  to  be  used  in  bacteriological  work  must  be  clean; 
that  is,  free  from  all  extraneous  organic  as  well  as  inorganic  matter.  To 
accomplish  this,  it  is  necessary  to  use  an  abundance  of  pure  water,  hot  as 
well  as  cold,  aided  by  sand,  paper  shreds,  brushes,  towels,  alcohol,  acids, 
soap,  sodic  and  potassic  hydroxides,  and  whatever  else  may  be  necessary. 
Boil,  wash,  rinse,  and  wipe  within  and  without  repeatedly  until  it  looks,  and 

36 


BACTERIOLOGICAL    TECHNIC. 


37 


is,  absolutely  clean.     The  following  solution  will  be  found  useful  as  a  cleans- 
ing agent  for  old  as  well  as  new  glassware: 

Potassium  Bichromate,  6  parts. 

Sulphuric  Acid,  30  parts. 

Water,  40  parts. 

Of  course,  the  sulphuric  acid  must  be  added  little  by  little  with  constant 
stirring,  in  order  to  avoid  excessive  heat  development.     Soak  the  glassware 


FIG.  12. — Wire  baskets  for  holding  test-tubes.  Cylindrical  form  and  square  form. 
Each  basket  holds  about  fifty  test-tubes.  The  wire  is  galvanized  to  prevent  rusting.  The 
round  wire  baskets  should  be  used. 

in  this  solution  for  some  time,  several  hours  or  more,  and  rinse,  wash,  drain, 
and  wipe  thoroughly  afterward.     The  sole  object  to  be  attained  is  cleanliness 
in  the  true  sense  of  the  word.     The  glassware  must  be  clean  bacteriologically 
and  chemically;  that  is,  it  must  be  free  from  mi- 
crobes and  chemical  substances. 


2.  Plugging  Containers  with  Cotton. 

After  the  thorough  cleansing  above  outlined, 
the  test-tubes  and  flasks  are  plugged  with  a  good 
quality  of  non-absorbent  commercial  cotton.  The 
dry  cotton  plug  forms  a  most  efficient  germ  filter.  All 
microbes  are  caught  and  held  in  the  meshes  of  the 
cotton,  and  yet  the  air  is  permitted  to  pass  through 
into  the  tube  or  flask. 

Open  a  roll  of  cotton,  find  the  free  end,  and  lay  it 
out  on  the  work  table.  Take  the  test-tube  in  the  left 
hand;  remove  a  goodly  tuft  of  cotton  with  right 
hand,  using  thumb  and  first  and  second  fingers. 
Place  this  over  the  mouth  of  the  tube  or  flask,  and 
push  it  down  to  a  distance  of  1/2  to  3/4  inch 
glass  rod  rounded  (by  heat)  at  the  ends.  The 


Fig.  13. — Wire  basket 
filled  with  test-tubes  plug- 
ged with  cotton.  A  little 
cotton  should  be  placed 
in  the  bottom  of  the  bas- 
ket to  lessen  the  danger  of 
breaking  the  test-tubes. 
(Williams.) 

by    means  of  a  solid 
rod   must    not   be  too 


38  PHARMACEUTICAL    BACTERIOLOGY. 

thick,  as  it  will  then  not  permit  enough  cotton  to  enter  the  opening  n  or 
yet  too  thin,  as  it  will  then  be  forced  through  the  cotton.  The  plug  must 
not  be  too  tight,  as  that  would  interfere  with  subsequent  manipulations  nor, 
yet  too  loose,  for  obvious  reasons.  Enough  cotton  should  project  above  the 
opening  to  permit  of  ready  grasping  between  the  fingers  in  the  later 
operations. 

Plugging  may  also  be  done  with  fingers  alone,  but  this  is  tedious  and  non- 
professional.     A  far  better  method  is  to  use  a  pair  of  fairly  large  blunt- 


FIG.  14.— A  hot  air  sterilizer.  These  sterilizers  are  double-walled,  on  stand,  with  per- 
forations at  top  for  thermometers.  Ordinary  baking  ovens  which  can  be  secured  from 
hardware  dealers  will  serve  the  purpose. 

pointed  pincers.     Remove  the  cotton  from  the  roll  by  means  of  the  pincers 
and  insert  it  into  the  test-tube  with  the  pincers. 

Whatever  method  is  used,  remove  the  amount  of  cotton  required  to  plug 
one  tube  or  flask  at  one  time.  Do  not  attempt  to  plug  with  several  small 
pieces.  If  an  excess  of  cotton  projects  above  the  opening,  pluck  it  away 
with  the  fingers;  do  not  cut  it  away  with  scissors.  Plug  the  tubes  as 
uniformly  as  possible. 

3.  Filling  Test-tubes  with  Culture  Media. 

The  rule  is  to  pour  the  culture  media  hot,  although  this  is  not  absolutely 
essential.  For  example,  if  the  media  are  liquid  in  the  cool  or  cold  state,  as 
bouillon,  serum,  milk,  etc.,  they  may  be  poured  cold.  A  good  rule  is  to  pour 
a  desired  amount  of  the  media  just  as  soon  as  they  are  prepared,  whether 
they  are  still  hot  or  merely  warm  or  cold.  Of  course,  gelatin  and  agar 
media  must  be  poured  hot  or  must  be  liquefied  before  they  can  be  poured. 


BACTERIOLOGICAL   TECHNIC. 


39 


Fill  a  small  to  medium-sized  beaker  about  two-thirds  full  of  the  culture 
medium.  Grasp  a  plugged  tube  near  the  upper  end,  holding  it  between 
thumb  and  first  two  fingers  of  the  left  hand.  Remove  the  cotton  plug  by 
means  of  the  first  and  second,  second  and  third,  or  third  and  fourth  fingers  of 
the  right  hand,  grasping  the  free  portion  of  the  plug  with  the  back  of  the 
fingers  toward  the  cotton.  Holding  the  tube  slightly  inclined  on  a  level 
with  the  mouth,  take  beaker  with  medium  in  right  hand  (at  the  same  time 
holding  the  cotton  plug  as  described),  see  that  the  beak  rests  lightly  upon 
and  projects  slightly  over  the  edge  of  the  tube,  and  pour,  at  the  same  time 
shifting  the  eyes  to  the  lower  end  of  the  tube  to  watch  the  filling  process. 
Fill  tubes  one-third  full.  Set  down  the  beaker  and  replace  the  cotton  plug. 
Place  the  filled  tubes  in  special  wicker  baskets,  with  a  little  cotton  at  the 
bottom  to  prevent  breaking.  Some  practice  is  necessary  in  order  to  pour 
so  that  none  of  the  liquid  comes  in  contact  with  the  upper  third  of  the  tube. 
This  must  be  avoided,  in  order  to  prevent  the  cotton  plug  from  sticking. 
Tubes  may  also  be  filled  from  funnel  with  rubber  hose,  stop-cock,  and  glass 
nib  attachment.  Occasionally  it  is  desirable  to  place  exact  amounts  of 
culture  media  in  the  tubes,  in  which  case  a  graduate,  a  burette,  a  pipette,  or 
other  convenient  measuring  device  may  be  used. 


FIG.  15. — Diagrammatic  sectional  view  of  Arnold  steam  sterilizer  illustrating  the  principle 
of  steam  formation,  circulation  and  condensation. 

4.  Sterilization  of  Culture  Media. 

All  culture  media  in  tubes  as  above  set  forth,  and  the  portions  remaining 
after  the  desired  number  of  tubes  are  filled,  must  be  considered  as  being 
contaminated  with  living  microbes  and  their  spores.  These  microbes  and 
spores  are  killed  by  the  sterilizing  process.  For  all  ordinary  purposes  the 


PHARMACEUTICAL    BACTERIOLOGY. 


discontinuous  or  fractional  method  answers  the  purpose  admirably.  Place 
the  test-tubes,  flasks,  and  other  cotton-plugged  containers  with  culture  media, 
in  a  steam  sterilizer  (Arnold  steam  sterilizer,  either  board  of  health  or  cylin- 
drical form;  or  kitchen  vegetable  cooker  or  steamer).  The  test-tubes  are 
placed  in  wire  baskets  (rectangular  or  cylindrical) .  These  several  containers 
with  culture  media  are  exposed  to  live  steam  for  about  thirty  minutes,  where- 
upon the  flame  is  turned  out,  and  if  convenient  the  containers  are  allowed 
to  remain  in  the  sterilizer.  Caution  must  be  observed  to  guard  against  con- 
densed steam  running  into  the  several  containers.  The  better  way  is  to 

remove  the  containers  and  place  them  in  an 
incubator  kept  at  a  temperature  of  20°  C.  In 
twenty-four  hours,  or  thereabouts,  steam  is 
again  applied  for  thirty  minutes.  This  is  re- 
peated a  third  time  on  the  second  day  after 
the  first  sterilization.  The  first  sterilization 
presumably  kills  most  of  the  vegetative  cells. 
During  the  first  interval  of  twenty-four  hours 
most  of  the  spores  present  develop  into  vege- 
tative cells,  which  are  killed  at  the  second 
sterilization.  Should  any  survive  the  second 
steaming,  they  are  sure  to  be  killed  during  the 
third  sterilization.  During  this  time  the  cotton 
plugs  have  not  been  removed.  The  media 
thus  fractionally  or  discontinuously  sterilized 
are  now  ready  for  use  in  making  microbic 
cultures,  or  they  may  be  set  aside  for  an  indef- 
inite period  of  time. 

It  is,  of  course,  evident  that  in  the  above 
process   of  sterilization   the  temperature  does 

FIG.  16.— Autoclave  for  using  not  exceed  ioo°  C.,  and  it  may  be  less  in  cer- 
steam    under    pressure    for    pur- 
poses of  sterilization.  tain    portions    of    the    sterilizer,    steamer,    or 

cooker,  say,  95°  to  97°  C.  In  large  or  well- 
equipped  bacteriological  laboratories  certain  kinds  of  sterilizations  are  done 
by  steam  under  pressure.  The  apparatus  used  for  this  purpose  is  known  as 
autoclave.  It  consists  of  a  strong  steam  cylinder  with  a  screwed-down  top 
safety  valve,  steam  gauge,  and  thermometer.  The  articles  (media,  etc.)  to 
be  sterilized  are  placed  inside,  the  top  is  securely  fastened  down,  steam  is 
generated  until  the  thermometer  registers,  say,  120°  C.  The  temperature 
is  kept  up  to  that  degree  for  about  five  to  ten  minutes,  which  is  sufficient  to 
destroy  all  life,  including  spores.  For  ordinary  purposes  the  autoclave  is 
not  essential.  In  fact,  its  use  is  rather  limited.  Blood  serum,  gelatin  media, 
and  all  media  containing  carbohydrates,  undergo  certain  chemical  changes 


BACTERIOLOGICAL   TECHNIC.  41 

when  the  temperature  is  raised  above  100°  C,  or  even  if  kept  at  100°  C.  for 
a  long  time  or  for  a  short  time,  if  oft  repeated.  The  autoclave  is  convenient 
for  sterilizing  discarded  cultures,  test-tubes,  and  glassware  generally,  and 
such  media  as  beef  broth  and  agar. 

In  many  instances  it  is  desirable  to  sterilize  at  a  temperature  lower  than 
100°  C.  Albumen  and  blood  serum,  for  instance,  will  coagulate  at  that 
temperature.  Again,  it  is  desired  to  kill  the  microbes  without  destroying 
the  toxins  which  they  form,  as  in  the  manufacture  of  bacterial  vaccines.  In 
the  sterilization  (pasteurization)  of  milk,  a  lower  temperature  is  employed. 
In  the  sterilization  of  these  and  other  substances  the  temperature  ranges 
from  50°  C.  to  85°  C.  The  discontinuous  method  is  employed,  differing 
from  the  method  already  described  in  that  the  period  of  exposure  is  much 
prolonged,  about  one  hour.  The  number  of  daily  exposures  ranges  from  one 
to  six.  For  example,  milk  exposed  to  a  temperature  of  60°  to  70°  C.  for  one 
hour  is  considered  sufficiently  sterilized,  whereas  blood  serum  is  subjected 
to  hourly  exposures  of  a  temperature  of  60°  C.  for  six  successive  days  before 
it  is  pronounced  completely  sterilized. 

5.  Preparation  of  Culture  Media. 

The  pharmacist  should  give  especial  attention  to  the  preparation  of 
bacterial  culture  media,  as  in  this  he  may  be  of  service  to  the  physician. 
The  busy  general  practitioner  who  is  not  eqipped  with  a  suitable  bacterio- 
logical laboratory,  or  who  does  not  have  time  to  prepare  culture  media,  would 
no  doubt  consider  it  a  very  decided  advantage  should  the  pharmacist  offer 
to  assist  him.  This  will  be  more  fully  set  forth  in  the  last  chapter. 

In  brief,  it  may  be  stated  that  microbes  feed  upon  the  same  substances 
that  we  feed  upon.  In  the  presence  of  adequate  warmth  and  moisture  they 
attack  all  organic  substances.  This  being  the  case,  it  may  readily  be  as- 
sumed that  there  are  many  substances  or  media  which  can  be  used  as  food 
for  bacteria.  Such  is  the  case,  and  the  number  of  media  which  have  been 
used  is  legion.  Almost  any  organic  substance  may  be  used,  provided  it  is 
not  aseptic  or  antiseptic  in  its  properties. 

Culture  media  are  liquid  or  solid,  simple  or  compound.  In  the  case  of 
liquid  or  liquefiable  solid  media,  the  following  physical  properties  are  de- 
sired, in  so  far  as  it  is  possible  to  attain  them: 

a.  Culture  media  should  be  perfectly  clear.     There  should  be  no  sedi- 
ment, no  opacity  or  flocculent  suspension,  and  no  floating  matter.    In  the  case 
of  broths,  extracts  generally,  gelatin  media,  and  blood  serum,  these  require- 
ments are  easily  attained.     Perfectly  clear  agar  is  difficult  to  obtain.     Milk 
is  normally  opaque. 

b.  Media  should  be  neutral  or  very  slightly  alkaline  to  litmus,  which  is 


PHARMACEUTICAL   BACTERIOLOGY. 


equivalent  to  a  slightly  acid  reaction  to  phenolphthalein,  at  a  temperature  of 
about  20°  C.     Most  microbes  develop  best  in  media  of  such  reaction. 

c.  They  must  be  free  from  living  microbes  and  their  spores,  and  from 


FIG.  17. — Arnold  Steam  Sterilizer.  Boston  Board  of  Health  Form.  This  sterilizer 
is  square,  and  constructed  with  a  side-door  all  in  accordance  with  the  recommendation  of 
the  Boston  Board  of  Health.  Its  large  size  makes  it  well  suited  to  the  requirements  of 
Board  of  Health  laboratories,  and  it  has  been  found  to  be  very  serviceable  and  convenient. 
It  is  made  of  copper  throughout,  following  the  same  principles  as  employed  in  the  construc- 
tion of  the  other  sterilizers. 


FIG.  18. — Arnold  Steam  and  Hot-Air  Sterilizer  for  Surgical  Instruments.  This 
sterilizer  is  a  combination  and  portable  sterilizer,  so  designed  that  instruments  may  be 
both  sterilized  and  then  dried  by  hot  air,  if  desired.  About  100°  C.  can  be  attained  with 
the  hot  air  by  simply  turning  the  valve  shown  in  the  illustration,  which  turns  the  steam  as 
it  escapes  from  the  chamber  into  the  base. 

other  organism.  This  requirement  is  attained  by  sterilization  as  already 
described.  Culture  media  contaminated  with  living  organisms  are  not 
usable  in  bacteriological  work. 


BACTERIOLOGICAL    TECHNIC. 


43 


The  essential  requirements  given  under  a,  b,  and  c  are  obtained  by  nitra- 
tion, neutralization,  and  sterilization,  as  will  be  more  fully  explained.  Non- 
liquefiable  solid  media,  as  potato,  bread,  squash,  etc.,  must  be  clean,  free 
from  living  microbes  and  other  organisms,  and  there  should  be  a  compara- 
tively smooth  exposed  inoculating  surface.  These  requirements  are  attained 
by  washing  and  otherwise  cleansing,  disinfecting,  rinsing,  and  heat  sterili- 
zation (dry  heat,  steam  or  hot- water  bath). 

The  following  are  the  more  important  media: 

A.  Nutrient  Bouillon. — 

Beef  Extract  (Armour's,  Liebig's,  etc.),  3  gm. 

Peptone,  10  gm. 

Salt,  5  gm. 

Distilled  Water,  1000  c.c. 

Mix  ingredients  and  boil  for  a  few  minutes.  Filter 
through  filter  paper.  This  bouillon  may  be  modified 
by  adding  glycerin  (6  per  cent.)  and  sugars,  as  dextrose, 
saccharose,  or  lactose  (i  per  cent.). 

B.  Loeffler's   Blood  Serum. — Very   largely   used  in 
making    diagnostic    diphtheria    bacillus   cultures.     In 
many  cities  this  medium,  with  sterilized  cotton  swabs, 
in  sterilized  test-tubes,  is  furnished  free  to  physicians 
by  the  board  of  health.     In  cities  and  towns  where  this 
is  not  done,  the  pharmacist  should  be  prepared  to  fur- 
nish the  materials  to  the  physicians.     The  medium  con- 
sists of 


Bouillon  with  i  per  cent.  Glucose, 
Blood  Serum, 


i  part. 
3  parts. 


FIG.  19. — Culture 
tube  and  swab  tube 
used  by  physicians  in 
the  diagnosis  of  diph- 
theria. The  swab 
tube  should  be  long 
enough  to  have  the 
entire  length  of  swab 
inside,  not  projecting 
as  shown  in  the  fig- 
ure. (Williams.} 
The  bouillon  is  prepared  as  above  described,  with  i 

per  cent,  of  glucose  added.  The  blood  serum  can  be  obtained  from  calf,  sheep, 
ox,  or  cow,  through  the  butcher  or  at  the  abattoir.  Collect  the  blood  in  a  clean, 
sterile  jar  or  flask,  closed  with  cotton  plug.  Place  on  ice  for  twenty-four  to 
forty-eight  hours,  during  which  time  coagulation  has  taken  place;  the  serum 
may  then  be  siphoned  off.  The  proper  sterilization  of  Loeffler's  serum  re- 
quires care.  After  the  bouillon  and  serum  are  mixed,  pour  into  test-tubes 
and  coagulate  in  a  Koch-serum  coagulator  at  a  temperature  of  80°  C.  Any 
form  of  sterilizer  may,  however,  be  used.  The  essentials  are  that  the  tern: 
perature  should  be  raised  very  gradually  and  must  be  kept  below  the  boil- 
ing-point, and  the  tubes  should  be  slanted  at  a  degree  which  will  bring  the 
medium  close  to  the  cotton  plug,  making  what  are  commonly  called  tube 
slants.  After  the  medium  is  coagulated  in  the  tubes  it  is  sterilized  frac- 


44  PHARMACEUTICAL    BACTERIOLOGY. 

tionally  on  three  successive  days  (one  hour  each  day)  at  a  temperature  of 
80°  C.  These  tube  slants  are  now  ready  for  the  physician. 

To  prevent  evaporation  of  the  medium  in  the  test-tubes,  cover  the  cotton 
plug  and  upper  end  of  tube  with  tin  foil  fastened  with  thread,  and  dip  into 
melted  paraffin  several  times.  Tubes  thus  sealed  can  be  kept  for  a  year  or 
more  without  any  considerable  shrinking  of  the  medium.  Dip  the  tin  foil  in 
a  1:2000  corrosive  sublimate  solution  before  capping  on  tubes. 

A  simpler  way  is  to  use  rubber  caps  which  are  especially  made  to  fit  over 
the  end  of  the  test-tube  and  the  cotton  plug.  These  rubber  caps  must  be 
sterilized  before  applying  them,  for  which  purpose  the  1-2000  corrosive 
sublimate  solution  will  be  found  satisfactory.  Rubber  stoppers  may  also  be 
used  but  they  are  more  expensive  and  inferior  to  the  rubber  cap  or  the  tin 
foil  with  coat  of  paraffin. 

C.  Liquid  Blood  Serum. — Obtained   as  for  Loeffler's  serum.     Sterilize 
fractionally  at  a  temperature  of  from  56°  to  58°  C.  for  one  hour  on  each  of 
six  days.     The  serum  will  be  liquid  and  clear. 

D.  Milk. — Secure  fresh  milk  directly  from  cow,  or,  if  in  cities,  demand 
certified  milk.     Keep  on  ice,  in  a  covered  jar,  for  twenty-four  hours.     Siphon 
off  the  middle  portion,  rejecting  cream  and  sediment.     Sterilize  like  Loeffler's 
blood  serum.     Litmus  milk  is  prepared  by  adding  i  per  cent,  of  azolitmin 
before  sterilizing.     This  indicator  will  show  whether  or  not  acids  are  formed 
by  the  microbes  which  may  be  cultivated  in  the  milk.     Only  pure  milk  will 
answer  the  purpose.     Milk  to  which  preservatives  (formaldehyd,  salicylic 
acid,  borax,  boric  acid)  have  been  added  must  not  be  used. 

E.  Peptone  Solution. — The  medium  is  employed  to  test  for  the  develop- 
ment of  indol  by  certain  bacteria.     It  consists  of 

Peptone,  10  gm. 

Salt,  5  gm. 

Distilled  Water,  1000  c.c. 

Boil,  filter,  and  sterilize  as  for  bouillon.  The  bacteriological  indoF  test 
is  of  great  importance  in  medical  practice,  and  the  chances  are  that  physi- 
cians will  require  this  medium.  However,  sugar-free  beef  broth  is  also  used 
for  this  test;  in  fact,  it  is  generally  preferred.  Beef  contains  a  small  amount 
of  muscle  sugar,  which  must  first  be  removed. 

F.  Sugar-free  Bouillon. — Grind  the  fat-free  beef  through  a  meat  grinder; 
add  water,  and  inoculate  at  once  with  a  pure  culture  of  Bacillus  coli  communis, 
and  allow  to  incubate  for  twelve  to  fifteen  hours  at  38°  C.,  then  boil,  filter, 
add  peptone  and  salt,  and  prepare  like  bouillon;  or,  inoculate  nutrient 
bouillon  with  the  colon  bacillus  and  prepare  as  above.     However,  before 
using  the  medium  it  should  be  tested  for  indol,  as  it  has  been  proved  that 
B.  coli  communis  may  form  indol  in  beef  extract.     The  indol  test  in  bacterial 
cultures  is  made  by  adding  two  drops  of  concentrated  sulphuric  acid  and  one 


BACTERIOLOGICAL   TECHNIC. 


45 


drop  of  a  o.oi  per  cent,  sodium  nitrite  solution  to  a  four-day  peptone-broth 
culture.  If  a  pink  color  appears  at  the  end  of  one-half  hour  it  indicates  the 
presence  of  indol. 

G.  Beef  Broth. — This  medium  is  now  not  as  extensively  used  as  formerly. 
It  is  more  difficult  to  prepare,  and  shows  no  advantages  over  the  bouillon 
already  described. 

Ground  or  Chopped  Lean  Beef,  500  gm. 

Peptone,  10  gm. 

Salt,  5  gm. 

Distilled  Water.  1000  c.c. 

Add  the  water  to  the  minced  meat,  shake  frequently,  and  keep  on  ice  for 
twenty-four  hours,  then  strain  forcibly  through  cloth,  or  press  out  in  a  hand 

press.  Add  the  salt  to  the  liquid,  boil,  make 
up  to  1000  c.c.,  and  add  the  peptone.  Neu- 
tralize, filter,  and  sterilize.  It  will  be  ap- 
parent that  the  cold  water  meat  infusion 
contains  merely  the  meat  salts,  meat  sugar, 
and  acids,  and  a  certain  proportion  of  the 
albumens.  The  albumens  are  coagulated 
and  removed  in  the  filtering  process,  so  that 
nothing  remains  of  the  meat  but  the  salts, 
acids,  and  the  trace  of  muscle  sugar.  Nearly 
the  whole  of  the  meat  proper  is  wasted.  It 


FIG.  20.  FIG.  21. 

FIG.  20. — Test-tube  cultures,  a,  Stab  culture.  This  tube  is  closed  with  a  rubber 
stopper  to  prevent  drying  of  medium ;  b,  streak  or  smear  culture  on  slant,  tube  closed  with 
rubber  cap.  (Williams?) 

FIG.  21. — The  ordinary  rice  cooker.  A  most  valuable  apparatus  in  preparing  culture 
media  and  for  sterilizing  test-tubes  and  other  objects. 

is  apparent,  therefore,  that  the  meat  extract  bouillon  answers  all  the  pur- 
poses of  the  beef  broth. 
H.  Gelatin  Medium. — 

Beef  Extract,  3  gm. 

Gelatin,  100  gm. 

Salt,  5  gm. 

Peptone,  10  gm. 

Distilled  Water,  1000  c.c 


46 


PHARMACEUTICAL   BACTERIOLOGY. 


Mix  ingredients  in  a  rice  cooker  and  boil  for  one-half  hour,  stirring  fre- 
quently; neutralize  and  filter.  This  forms  a  very  efficient  culture  medium 
for  most  bacteria,  and  is  clear  and  remains  solid  at  ordinary  temperatures. 
It  must  be  borne  in  mind,  however,  that  frequent  or  prolonged  heating  tends 
to  liquefy  gelatin  permanently. 


FIG.  22. — This  is  a  copper  double-walled  incubator  covered  with  non-conducting 
material  and  provided  with  a  water  gauge,  tubulations  for  thermometer  and  thermostat, 
a  ventilating  strip,  enclosed  base  and  inner  glass  door.  The  incubating  chamber  is  24  cm. 
high,  30  cm.  wide  and  24  cm.  deep. 


I.  A  gar  Medium. — Agar  is  a  seaweed  found  on  the  Japanese  coast.  It 
forms  an  important  article  of  diet  among  the  Japanese  and  Chinese.  The 
medium  consists  of 


Beef  Extract, 

Agar, 

Salt, 

Peptone, 

DistiUed  Water, 


3  gm. 

J5  gm- 

5  gm. 

10  gm. 

1000  c.c. 


Prepare  like  the  gelatin  medium.  Agar  is  difficult  to  filter,  and  the 
medium  is  never  quite  clear.  The  agar  medium  liquefies  at  a  higher  tem- 
perature than  gelatin,  and  does  not  tend  to  remain  liquid,  no  matter  how 
often  or  how  long  it  may  be  heated. 


BACTERIOLOGICAL   TECHNIC. 


47 


J.  A  gar-gelatin  Medium. — This  has  the  advantage  of  both  media,  and  is 
now  much  used  in  general  bacteriological  work. 


Agar, 
Gelatin, 
Salt, 
Peptone, 
Distilled  Water, 


8  gm. 
40  gm. 

5  gm. 
10  gm. 

IOOO    C.C. 


Mix,  boil  in  rice  cooker,  stir;  neutralize,  filter,  and  sterilize  as  for  other 
media. 

The  above  includes  the  more  important  culture  media  used  in  bacteriolog- 
ical work.  Others  can  be  prepared  as  occasion  requires.  It  is  not  neces- 


FIG.  23. 


FIG.  24. 


FIG.  23. — MurrilPs  Gas  Pressure  Regulator.  This  apparatus  in  its  most  improved 
form  is  to  be  used  in  connection  with  a  thermostat  for  the  maintenance  of  a  constant 
emperature.  The  use  of  this  regulator  relieves  the  thermostat  of  the  necessity  of  caring 
for  the  wide  viriation  which  is  apt  to  occur  in  the  gas  pressure,  and  with  it  the  temperature 
may  be  held  constant  to  within  0.1°  C. 

FIG.  24. — Reichert  thermo-regulator  or  thermostat  used  with  incubator  and  other 
apparatus  requiring  a  uniform  degree  of  temperature.  May  be  used  in  conjunction  with 
the  gas  pressure  regulator. 

sary  to  make  up  the  full  amounts  indicated  if  it  is  evident  that  smaller 
quantities  will  suffice.  The  student  should  prepare  all  of  the  media  in  small 
amounts  (one-quarter  the  quantities  given)  several  times,  in  order  to  get 
the  necessary  experience  and  practice. 

6.  General  Directions  for  the  Preparation  of  Culture  Media. 
Book  information  alone  is  not  sufficient.     Experience  must  be  added. 


48  PHARMACEUTICAL    BACTERIOLOGY. 

Also,  brief,  concise  explanations  are  far  more  valuable  than  lengthy  descrip' 
tions  of  unessential  details.  Those  possessed  of  good  judgment  do  not  re- 
quire lengthy  explanations,  and  lengthy  explanations  would  certainly  be 
wasted  on  those  who  lack  good  judgment.  This  does  not  imply,  however, 
that  it  is  unnecessary  to  adhere  strictly  to  established  methods.  The  novice 
must  follow  closely  the  methods  formulated  by  those  who  have  devoted  many 
years  to  some  one  particular  mode  of  procedure,  as  it  is  wholly  unlikely 
that  he  can  improve  upon  them.  Furthermore,  when  a  physician  calls 
for  Loeffler's  blood  serum,  for  example,  he  wishes  to  be  assured  that  the 
medium  has  been  prepared  according  to  the  standard  method.  Any  sub- 
stitution or  deviation,  no  matter  how  slight,  may  bring  about  wholly  neg- 
ative or  erroneous  results  and  conclusions.  With  this  in  mind  the  follow- 
ing suggestions  are  added: 

A.  Selection  of  Ingredients. — Great  care  must  be  observed  in  the  selection 
of  the  ingredients  used  in  the  preparation  of  culture  media.     Meats  used 
must  be  from  healthy  animals,  and  there  must  be  absolute  certainty  that  no 
preservative  has  been  added.     Buy  the  meat  personally  from  the  nearest 
reliable  butcher  who  keeps  fresh  meats  only.     Remove  as  much  of  the  fat 
as  possible.     The  so-called  round  steak  of  beef  is  usually  employed. 

Use  only  the  best  gelatin;  the  so-called  best  French  gelatin  is  usually 
employed,  although  much  of  the  " French  gelatin"  comes  from  Berlin, 
Chicago,  Omaha,  or  other  places  equally  remote  from  France.  Do  not 
attempt  to  use  old  friable  gelatin. 

The  milk  requirements  have  already  been  referred  to.  The  milk  must 
be  fresh,  placed  on  ice  at  once,  and  sterilized  within  twenty-four  hours 
after  it  is  taken  from  the  cow.  If  the  milk  is  obtained  from  an  unknown 
dealer,  test  it  for  the  presence  of  added  water,  preservatives,  and  other 
foreign  matter. 

Agar  does  not  deteriorate  readily,  and  may  be  kept  in  good  condition 
for  a  long  time.  Other  highly  gelatinous  seaweeds  may  be  used,  although 
this  is  not  permissible  in  the  preparation  of  any  of  the  standard  culture 
media. 

Serum,  egg  albumen,  peptone,  various  indicators,  etc.,  must  be  pure. 
Too  much  caution  cannot  be  observed  in  this  regard.  Secure  the  blood  for 
serum  personally  wherever  possible,  from  healthy  animals.  Use  egg  albu- 
men from  fresh  eggs,  not  from  cold-storage  eggs.  Peptone  and  other  chem- 
icals should  be  secured  from  reliable  dealers. 

B.  Suggestions  on  the  Preparation  of  Culture  Media. — First  of  all,  some 
experience  is  necessary  before  a  neat  article  can  be  prepared.     Do  not 
expect  to  prepare  a  medium  which  meets  all  of  the  requirements  the  very  first 
time.     In  preparing  gelatin  media,  remember  that  these  are  injured  by  excess- 
ive heating,  and  in  preparing  agar  media,  remember  that  they  are  very  difficult 


BACTERIOLOGICAL    TECHNIC.  49 

to  filter.  Both  must  be  filtered  hot,  using  hot-water  funnels;  or  the  ordinary 
filtering  device  can  be  used  by  keeping  the  unfiltered  portion  hot  and  pouring 
into  the  funnel  from  time  to  time.  Cover  funnel  with  filter  paper  to  keep  out 
dust,  and  keep  in  the  heat  as  much  as  possible.  In  so  far  as  possible  filter  all 
media  through  filter  paper  (one  thickness,  properly  folded),  but  it  is  practi- 
cally impossible  (for  reasons  of  time)  to  pass  agar  through  filter  paper.  This 
medium  is  usually  filtered  through  cotton  upon  which  a  neatly  folded  and 
perforated  sheet  of  filter  paper  has  been  placed.  Puncture  the  filter  paper 
several  times  with  a  small  knife  blade.  Filtering  through  cotton  is  quick, 
but  the  media  are  much  less  clear  than  when  filtered  through  filter  paper. 
The  filtering  process  may  also  be  hastened  by  means  of  pressure  (suction) ; 
connect  funnel  with  aspirator  bottle  and  pump,  but  see  to  it  that  the  connec- 
tions with  the  hydrant  are  properly  made  and  that  the  flow  is  properly  reg- 
ulated, in  order  to  guard  against  any  back  pressure,  which  may  cause  the 
receiver  to  fill  with  hydrant  water.  This  accident  is  best  avoided  by 
interpolating  a  flask  or  bottle.  Agar  may  also  be  clarified  by  pre- 
cipitation. Pour  the  hot  agar  into  an  ordinary  percolator  used  by  phar- 
macists. The  dirt  particles  and  other  impurities  will  gradually  settle  to  the 
bottom.  When  cool,  take  out  the  solid  medium  and  cut  away  the  lower 
portion  cantaining  the  sediment. 

C.  Neutralization  of  Culture  Media. — As  already  stated,  most  bacteria 
grow  best  in  neutral  or  very  slightly  alkaline  (to  litmus)  media,  and  since 
most  media  are  quite  decidedly  acid  in  reaction,  it  is  desirable  to  alkalinize. 
This  is  done  by  means  of  normal  sodium  hydroxide  solution.  In  order  to 
understand  the  method  of  procedure  clearly,  it  is  necessary  to  make  certain 
explanations. 

A  normal  (N/i)  solution  of  any  substance  contains  as  many  grams  per 
liter  of  the  substance  as  there  are  units  in  its  molecular  weight,  if  the  substance 
contains  one  atom  of  replaceable  hydrogen.  If  it  contains  two  atoms  of 
replaceable  hydrogen,  the  number  of  grams  used  equals  the  molecular 
weight  divided  by  two,  and  so  on.  According  to  this,  a  normal  solution  of 
sodium  hydroxide  contains  40  gm.  of  sodium  hydroxide  in  a  liter. 
Exact  normal  solutions  are,  however,  not  prepared  by  weight.  Crystallized 
oxalic  acid  is  used  as  the  basis  for  making  normal  solutions.  This 
acid  has  a  molecular  weight  (including  a  molecule  of  water  of  crystallization) 
of  126,  and,  since  it  is  dibasic,  63  gm.  per  liter  are  taken.  Any  normal 
acid  solution  will  exactly  neutralize  an  equal  volume  of  normal  alkaline 
solution.  To  make  a  normal  sodium  hydroxide  solution,  add  about  41  gm. 
of  pure  caustic  soda  to  one  liter  of  distilled  water.  Determine  the  amount 
of  this  solution  required  to  just  neutralize  i  c.c.  of  normal  oxalic  acid 
solution.  This  volume  contains  the  quantity  of  sodium  hydroxide  which 
should  be  present  in  i  c.c.  of  normal  solution,  and  from  this  we  may 

4 


50  PHARMACEUTICAL    BACTERIOLOGY. 

calculate  the  volume  of  distilled  water  to  be  added  in  order  that  i  c.c.  of 
sodium  hydroxide  solution  will  neutralize  i  c.c.  of  normal  oxalic  acid  solution. 
Having  a  normal  solution  of  sodium  hydroxide,  it  is  now  possible  to 
prepare  a  normal  solution  of  hydrochloric  acid,  etc.  A  tenth-  (N/io), 
twentieth-  (N/2o),  fiftieth-  (N/5o)  normal  solution  is  a  normal  solution 
diluted  ten,  twenty,  and  fifty  times. 

An  acid  reaction  is  indicated  by  + ,  and  an  alkaline  by  — .  The  degree 
of  acidity  of  any  culture  medium  in  preparation  may  be  indicated  by  the 
amount  of  normal  sodium  hydroxide  solution  required  to  render  it  neutral 
to  phenolphthalein.  Neutralization  by  titration  is  done  as  follows:  Place 
5  c.c.  of  the  medium  to  be  neutralized  in  a  dish,  add  45  c.c.  of  distilled 
water,  stir,  and  bring  to  a  boil.  Add  i  c.c.  of  phenolphthalein  solution 
(0.5  per  cent,  of  phenolphthalein  in  50  per  cent,  alcohol).  Add  enough 
of  twentieth-normal  sodium  hydroxide  solution  (in  a  burette),  with  constant 
stirring,  to  give  a  faint  but  distinct  pink  color.  Read  the  amount  of  twentieth- 
normal  sodium  hydroxide  necessary  to  neutralize  the  5  c.c.  of  medium,  and 
from  this  calculate  the  amount  of  normal  sodium  hydroxide  solution  neces- 
sary to  neutralize  the  entire  quantity  of  culture  medium.  Now  boil  the 
medium,  and  again  titrate,  when  it  will  be  found  that  there  is  a  slight 
acid  reaction.  A  third  titration  is  rarely  necessary. 

Another  method  is  to  take  10  c.c.  of  the  culture  medium,  add  a  few  drops 
of  the  phenolphthalein  solution.  From  a  burette  add,  drop  by  drop,  with 
constant  stirring,  a  normal  sodium  hydroxide  solution  (0.4  per  cent.)  until  a 
faint  pink  color  appears,  which  indicates  the  beginning  of  the  alkaline  reac- 
tion. Repeat  this  with  two  more  samples.  Note  the  amount  of  sodium 
hydroxide  solution  required  in  each  case,  and  take  the  average  and  calcu- 
late the  amount  required  for  the  entire  quantity  of  medium.  If,  for  example, 
the  average  was  i  c.c.  for  each  10  c.c.  of  medium,  then  1000  c.c.  of  bouillon 
would  require  100  c.c.  of  the  sodium  hydroxide  solution;  a  concentrated 
solution  being  used,  in  order  to  avoid  the  dilution  of  the  medium  with  the 
water  of  the  caustic-soda  solution.  Flocculency  of  the  medium  usually 
indicates  excessive  alkalinity. 

The  old,  crude,  rough-and-ready  method  is  to  add,  from  a  beaker, 
drop  by  drop,  a  tenth-normal  sodium  hydroxide  solution,  with  constant  stir- 
ring, until  red  litmus  paper  just  begins  to  turn  blue.  In  practice  it  is  found 
that  when  a  culture  medium  is  neutral  or  slightly  alkaline  to  litmus  it  is  still 
acid  to  phenolphthalein.  In  fact,  it  is  claimed  that  most  bacteria  develop 
best  in  a  medium  having  a  reaction  indicated  by  + 1  or  +0.5;  that  is,  it  is 
sufficiently  acid  to  phenolphthalein  to  require  i  per  cent,  or  0.5  per  cent,  of 
normal  sodium  hydroxide  solution  to  render  it  neutral  to  phenolphthalein. 

D.  Suggestions  on  the  Preparation  of  Culture  Media  for  Physicians. — First 
of  all,  the  pharmacist  must  have  the  necessary  laboratory  equipment  and 


BACTERIOLOGICAL   TECHNIC.  51 

necessary  skill  and  experience  to  prepare  culture  media.  He  should  explain 
to  a  few  representative  physicians  that  he  is  ready  to  prepare  such  media  as 
the  busy  physician  may  require.  The  physicians  will  in  all  probability 
indicate  what  media  are  likely  to  be  needed  in  the  course  of  their  practice. 
Allow  yourself  to  be  guided  by  these  several  suggestions  and  prepare  the 
media  accordingly. 

Make  sure  that  the  culture  media  are  clear.  There  must  be  no  sediment 
and  no  flocculency.  Not  infrequently  the  medium  fails  to  become  sufficiently 
clear,  even  though  every  precaution  has  been  taken.  In  such  cases  clari- 
fication may  be  tried,  rather  than  to  discard  it.  Add  the  white  of  an  egg,  thor- 
oughly beaten,  to  a  liter  of  the  medium  in  the  liquid  state  and  at  a  tempera- 
ture below  the  coagulating  point  for  albumen,  mix 
thoroughly;  boil  for  ten  minutes,  and  filter.  The 
coagulating  albumen  takes  up  the  impurities  which 
remain  upon  the  filter  with  the  albumen,  while  the 
medium  comes  through  perfectly  clear.  Media  which 
have  become  infected  with  bacteria  as  the  result  of 
inadequate  setrilization  should  be  discarded.  Do  not 
attempt  to  clarify  them.  They  may  become  clear, 
but  they  are  nevertheless  objectionable  because  of  the 
substances  which  the  bacteria  may  have  liberated  and 

which  might  interfere  with   the  development  of  the  ,   FIG.  25.— Koch  safety 

burner.       Should     the 
bacteria  to  be  grown  in  it  subsequently.  flame  be  blown  out,  an 

Most  of   the  tubes   with    solid   media    (Loefifler's  automatic  devise  shuts 

.  1111°"  the  8as- 

serum,    gelatin,    agar,  and    gelatme-agar)    should  be 

slants.  The  slanting  surface  offers  certain  advantages  in  making  diag- 
nostic bacterial  cultures.  The  usual,  non-slanting  tubes,  for  deep  stab 
cultures,  should,  however,  also  be  held  in  readiness.  Keep  all  tubes 
in  suitable  containers,  in  a  dry,  cool,  clean  place.  To  guard  against 
infection  by  mould  and  other  organisms,  it  is  well  to  cap  all  tubes  with  the 
rubber  caps  or  the  tin  foil  dipped  in  corrosive  sublimate  and  parafhn,  as 
already  suggested.  In  case  of  liquid  media,  the  rubber  stoppers  or  the 
rubber  caps  are  much  preferred,  or  the  hot  paraffin  may  be  painted  over  the 
tin  foil  and  upper  end  of  tube  by  means  of  a  small  brush.  Apply  two  or 
three  coats.  Thus  protected,  there  is  no  danger  of  outside  infection. 

The  chances  are  that  the  physician  who  calls  for  tube  culture  media  will 
also  require  the  use  of  an  incubator.  This  the  pharmacist  should  have  in 
readiness.  The  usual  copper  double-walled  water-jacket  incubator,  with 
thermo-regulator,  kept  at  a  temperature  of  about  25°  C.,  will  serve  the 
purpose. 

The  swab  to  be  supplied  with  each  tube  of  slanted  Loeffler's  serum 
consists  of  a  piece  of  wire  or  of  pine  wood  four  inches  long,  around  the 


52 


PHARMACEUTICAL   BACTERIOLOGY. 


lower  end  of  which  a  pledget  of  absorbent  cotton  has  been  wound  and 
firmly  tied  by  means  of  thread.  This  is  placed  in  a  test-tube,  which  is  then 
plugged  with  cotton  and  sterilized  in  the  dry  sterilizer  (one  hour  at  a  tempera- 
ture of  140°  C.)  The  physician  wipes  the  cotton  end  of  the  swab  over  the 
suspected  throat  area,  and  then  lightly  rubs  it  over  the  surface  of  the  serum 


FIG.  26. — Hot  water  funnel  with  stand 
and  ring  gas  burner. 


FIG.  27. — Hot  water  funnel 
with  stand. 


tube  'slant.  The  swab  is  returned  to  the  tube,  the  cotton  plug  is  restored 
and  then  returned  to  the  board  of  health  to  be  destroyed  in  stove  or 
furnace  fire,  or  destroyed  by  the  attending  physician  in  case  there  is  no 
board  of  health  to  recieve  it. 


FIG.  28. — Glass  rods  with  platinum  wire,  straight  and  loop,  for  inoculating  culture  tubes. 
Petri  plates,  etc.— (Williams.) 

7.  Making  Bacterial  Cultures. 

This  branch  of  the  science  of  bacteriology  is  of  comparatively  little 
importance  to  the  pharmacist.  While  it  is  desirable  to  know  what  bacterial 
cultures  are  and  how  to  make  some  of  them,  it  is  wholly  unlikely  that  the 
pharmacist  will  be  called  upon  to  do  extensive  work  along  this  line.  This  is 


BACTERIOLOGICAL   TECHNIC. 


53 


the  work  of  those  who  make  bacteriology  a  specialty.  Such  bacterial 
cultures  as  are  likely  to  come  to  the  notice  of  pharmacists  will  most  generally 
be  prepared  by  physicians,  health  officers,  and  other  specialists  in  bacteri- 
ology. The  pharmaceutical  bacteriologist  may  be  called  upon  to  make 
bacterial  examinations  of  drinking  water,  of  milk,  of  ice  cream,  and  other 
food  materials;  of  syrups,  liquors,  aquae,  tinctures,  fluidextracts,  infusions, 
etc.,  and  he  should,  if  possessed  of  some  skill  and  adequate  laboratory 
facilities,  be  able  to  do  so. 

The  prime  object  in  growing  bacteria  in  artificial  culture  media  is  to 
make  possible  their  further  more  careful  and  more  extended  study.     The 


FIG.  29. — Cover-glass  pincers. 


a  and  b  are  self-clamping  but  the  pressure  is  often  enough 
to  break  thin  covers. 


study  of  bacteria  in  their  natural  or  normal  surroundings  is  all-important, 
but  is  not  complete  without  the  artificial  culturing. 

As  a  rule,  bacteria  are  biologically  associated  with  other  organisms,  and 
it  is  unusual  to  find  pure  cultures  in  nature  or  in  natural  media.  An  open 
sore  may  contain  several  or  many  species  and  varieties  of  bacteria,  in  addi- 
tion to  the  pus  germs.  The  intestinal  tract  of  the  typhoid  patient  contains 
bacteria  other  than  the  comma  bacillus  of  Koch.  The  tubercular  bron- 
chials  always  show  a  mixed  infection.  The  diphtheric  membrane  con- 
tains some  foreign  germs,  etc.  Some  infections,  particularly  those  of 
internal  tissues  or  organs,  as  lymphatic  glands  for  example,  may  present 
practically  pure  cultures.  However,  no  matter  how  mixed  an  infection 


54 


PHARMACEUTICAL   BACTERIOLOGY. 


may  be,  there  is  always  a  predominating  type  present,  or,  to  state  it  more 
correctly,  it  is  the  unusual  development  of  the  predominating  type  which 
determines  the  diagnostic  characteristics  of  the  infection. 

It  must  also  be  borne  in  mind  that  bacteria  behave  differently  when 
taken  out  of  their  natural  environment  and  placed  in  artificial  culture  media. 
It  does  not  at  all  follow  that,  in  the  case  of  a  mixed  infection,  the  predominat- 
ing and  diagnostic  microbe  will  remain  the  predominating  type  when  said 
mixed  infection  is  transferred  to  some  artificial  culture 
medium.     In  fact,  the  predominating  microbe  may  develop 
very  slowly  or  with  great  difficulty,  if  at  all,  in  the  artificial 
culture  media;   whereas   one   or   more  of  the  associated 
microbes  may  thrive  remarkably  well,  soon  entirely  over- 
shadowing    the    former.     These    and    other    conditions 
occasion    some    of    the  great  difficulties  encountered  in 
determining  the   primary  causes  of  some  microbic  and 
protozoic  diseases  and  infections. 

A.   Test-tube    Cultures. — Inoculate    several    test-tubes, 
containing    nutrient    gelatin    or    agar   gelatin,  with  any 


FIG.  30. 
Fig.  30. — Cotton   plugged   tube  with  a  potato  slant   resting  on  a   bit  of  glass  rod   to 

keep  the  potato  out  of  the  water  in  the  bottom  of  the  tube.     (Williams.} 
FIG.  31. — Manner  of  holding  tubes  when  making  subcultures.     The  cotton  plugs, 
removed  from  the  two  tubes,  should  be  held  in  hand  holding  the  platinum  rod,  as  explained 
in  the  text.     (Williams.} 

material  which  is  known  to  be  bacterially  infected.  This  is  done  by 
touching  the  infected  material  with  the  tip  of  a  heat-sterilized  (by 
holding  in  flame  of  Bunsen  burner  until  red  hot)  platinum  needle  (pre- 
pared by  fusing  a  platinum  wire,  11/2  inches  long,  into  the  end  of  a 
glass  rod,  six  to  seven  inches  long),  then  removing  the  cotton  plug  from 
the  test-tube,  and  pushing  the  needle,  carrying  the  microbes,  into  the  culture 
medium  down  to  the  very  bottom  of  the  tube.  Replace  the  cotton  plug  at 
once,  pass  the  needle  into  the  flame  of  the  Bunsen  burner  until  red  hot,  to 
sterilize  it,  and  lay  aside  for  the  next  tube  inoculation.  This  is  known  as  a 
deep  stab  tube  inoculation.  In  this  manner  inoculate  some  five  or  six  tubes. 


BACTERIOLOGICAL   TECHNIC. 


55 


Also  make  streak  inoculation  in  tube  slants  by  simply  passing  the  infected 
platinum  needle  over  the  middle  of  the  tube  slant  surface,  from  lower  end 
toward  the  top,  observing  the  instructions  regarding  the  cotton  plug  and 
needle  sterilization,  with  each  tube  inoculation.  Number  the  tubes  seri- 
ally, and  in  a  special  notebook  make  entry  of  all  desirable  data  pertaining 
to  each  inoculation,  making  such  entries  under  each  tube  number.  Place 
tubes  vertically  in  a  suitable  holder,  as  tumbler,  beaker,  wire  basket,  etc., 
and  set  aside  in  incubator  or  in  some  container  to  which  you  alone  have 
access. 

In  warm  weather  the  first  bacterial  growths  may  appear  at  the  end  of 
thirty-six  hours.  In  cold  or  cool  weather  nothing  may  appear  for  two,  three, 
and  even  four  to  five  days.  Note  the  nature  of  the  bacterial  growth  in  a 
deep  stab  inoculation  and  in  the  streak  inoculation,  as  to 


FIG.  32. — Making  an  Esmarch  roll-tube  culture.  A  lump  of  ice  is  placed  in  a  dish 
and  the  inoculated  tube  is  placed  horizontally  in  a  groove  in  the  ice  and  revolved  until  the 
medium  is  well  set.  The  groove  may  be  made  with  test-tube  full  of  hot  water. 
(Williams.} 

a.  Growth — scanty,  moderate,  abundant,  slow,  rapid. 

b.  Form  of  growth — outline  clearly  defined,  spreading,  rugose,  beaded,  etc. 

c.  As  to  surface — flat,  raised,  concave,  convex. 

d.  Color — translucent,    glistening,    waxy,    transparent,    opaque,    light, 
chalky  white,  grayish-white,  dark  red,  green,  blue,  yellow,  lemon   color, 
purple,  etc. 

e.  Odor — comparative  description. 

f.  Consistency — viscid,    slimy,    stringy,  membranous,  friable  or  brittle, 
dry,  watery,  etc. 

g.  Changes  in  medium — gelatin  liquefied,  gelatin  not  liquefied;  colored, 
as  grayed,  browned,  reddened,  blued,  etc.     In  case  indicators  are  used,  the 
possible  color  changes  should  be  noted. 


50  PHARMACEUTICAL   BACTERIOLOGY. 

h.  Deep  stab  culture — where  is  growth  most  active?  If  at  bottom,  it 
indicates  anaerobic  tendencies.  If  limited  to  top  of  medium,  it  indicates  de- 
cidely  aerobic  tendencies.  (Most  bacteria  are  decidedly  aerobic;  that  is, 
they  require  oxygen  to  thrive.) 

The  test-tube  cultures  do  not  necessarily  represent  pure  cultures,  and 
the  student  cannot  know  whether  the  growths  in  the  test-tubes  represent  the 
predominating  bacterial  flora  in  the  substance  from  which  the  inoculations 
were  made.  The  chief  object  in  making  the  above  cultures  is  to  enable 
the  student  to  get  practice  in  this  preliminary  work,  particularly  as  to 
making  the  cultural  observations  above  indicated. 


FIG.  33. — Kitasato  filter  for  filtering  hypodermic  solutions,  culture  media,  sera,  water, 
etc.  The  material  to  be  filtered  is  placed  in  the  globose  container  and  forced  through  the 
clay  (infusorial  earth)  tube  (Berkefeld  filter  bougie)  by  connecting  the  receiver  with  a 
vacuum  pump.  All  parts  of  the  filter  must,  of  course,  be  sterilized  by  heat  before  and 
after  using.  (Williams.} 

The  student  should  now  make  transfers  (subcultures)  from  the  first  tube 
cultures  into  second  tubes,  and  note  whether  or  not  the  characteristics 
originally  noted  are  continued  or  repeated.  If  the  transfer  cultures  are  the 
same  as  the  originals,  it  is  an  indication  that  the  first  cultures  were  pure 
(representing  one  species  or  variety),  which  is  generally  the  case,  though 
it  must  be  borne  in  mind  that  one  and  the  same  species  of  microbe  may 
undergo  considerable  change  in  extended  culturing,  as  indicated  in  the 
changed  culture  characters.  In  fact,  some  of  the  changes  are  so  extreme  as 
to  confuse  even  the  most  expert  bacteriologists. 


BACTERIOLOGICAL   TECHNIC. 


57 


B.  Isolating  Bacteria  by  the  Plate  Method. — In  order  to  separate  or  isolate 
the  several  species  and  varieties  of  bacteria  in  any  contaminated  substance, 
it  is  only  necessary  to  dilute  the  inoculating  material  sufficiently.  For  this 
purpose  there  is  necessary,  sterilized  Petri  dishes  containing  heat-sterilized 
gelatin  or  other  solid  media  through  which  the  bacteria  from  the  contami- 
nated substance  are  disseminated  in  numbers  so  small  that  the  colonies  from 
each  and  every  microbe  present  may  be  visible  to  the  naked  eye  (or  aided 
by  a  simple  lens).  This  is  done  as  follows: 


FIG.  34. — Streak  culture  on  agar  in  a  Petri  dish.     (Delafteld  and  Prudden.) 


To  obtain  isolation  cultures  of  air  bacteria  it  is  only  necessary  to  expose 
the  Petri  dish  (with  a  layer  of  gelatin  or  agar-gelatin  medium,  sterilized)  for 
about  two  minutes,  immediately  closing  the  dish  and  setting  it  aside  to  await 
developments.  Making  isolation  cultures  from  contaminated  solids  or 
liquids  is  not  quite  so  simple.  Proceed  as  follows:  Liquefy  the  gelatin  in 
four  or  five  test-tubes  and  keep  them  at  a  temperature  of  not  more  than  30°  C., 


58  PHARMACEUTICAL   BACTERIOLOGY. 

just  high  enough  to  keep  the  contents  liquid;  set  them  in  a  beaker  filled 
with  warm  water  (30°  C.)  until  needed.  Number  the  tubes  from  i  to  5. 
Dip  a  platinum  loop  (bend  the  end  of  a  straight  needle  into  a  small  loop) 
into  the  infected  liquid,  as  bouillon,  milk,  water,  tea,  syrup,  tincture,  fluid- 
extract,  etc.,  etc.,  and  pass  one  loopful  into  tube  No.  i  (sterilize  loop  and 
return  to  its  proper  place).  Rotate  tube  (replugged  with  the  cotton  and  held 
vertically)  rapidly  between  the  hands  for  twenty  seconds,  to  mix  contents. 
By  means  of  the  platinum  loop  take  two  loopfuls  (one  loopful  may  serve) 
from  tube  No.  i  (which  you  have  just  inoculated  and  rotated)  and  pass 
them  into  tube  No.  2.  Plug  both  tubes,  set  aside  tube  No.  i,  and  rapidly 
rotate  tube  No.  2.  Take  two  loopfuls  from  tube  No.  2  and  transfer  to  tube 
No.  3,  and  proceed  as  before.  Now  pour  contents  of  tube  No.  i  into  a  sterile 


FIG.  35. — Appearance  of  colonies  on  gelatin  in  a  Petri  dish.  Differences  in  size  of 
colonies  may  indicate  different  species.  Differences  in  color  also  indicating  different 
species,  cannot  be  shown  in  the  figure.  (Williams.) 

Petri  dish,  also  numbered  i;  contents  of  tube  2  into  Petri  dish  2;  and  tube 
3  into  Petri  dish  3.  Wait  until  the  media  in  the  Petri  dishes  are  solidified, 
and  then  set  aside  at  the  room  temperature  to  await  developments.  In  the 
course  of  two  or  three  days  it  will  perhaps  be  found  that  very  many  minute 
specks  are  visible  in  dish  No.  i,  some  one  hundred  or  more  may  appear  in 
dish  No.  2,  and  perhaps  not  more  than  ten  or  twenty  in  dish  No.  3.  Observe 
carefully  the  several  growths  in  dishes  2  and  3.  Each  visible  growth  in- 
dicates the  development  from  a  single  microbe.  Are  the  several  growths 
all  alike,  or  do  they  differ?  Differences  in  color  and  in  outline  of  growths 
indicate  different  species  of  bacteria.  The  several  different  kinds  of  bacteria 


BACTERIOLOGICAL    TECHNIC. 


59 


may  now  be  transferred  to  test-tubes  by  means  of  the  straight  platinum  needle 
or  the  loop,  and  the  observations  may  thus  be  extended.  Transfers  can  be 
made  to  different  kinds  of  media,  as  agar,  gelatin,  agar-gelatin,  beef  broth, 
milk,  prepared  potato,  etc. 

C.  Making  Bacterial  Counts. — In  order  to  determine  the  number  of  bac- 
teria in  any  given  substance  the  same  procedure  as  was  just  described  is  fol- 
lowed, with  the  difference  that  a  definite  amount  of  the  thoroughly  mixed 
contaminated  substance  is  added  to  a  definite  amount  of  culture  medium  in 
the  test-tubes  in  which  the  dilution  mixtures  are  made.  For  example,  we  will 
suppose  that  it  is  desired  to  determine  the  number  of  bacteria  (per  c.c.)  in 
milk:  Thoroughly  mix  the  sample  of  milk  by  shaking  it  in  the  container. 
Take  o.i,  0.2,  0.5,  or  i  c.c.  of  the  milk  (by  means  of  a  sterilized  graduated 
pipette)  and  add  it  to  9  c.c.  of  the  liquefied  culture  medium  in  tube  No.  i; 
i  c.c.  of  tube  No.  i  to  tube  No,  2,  also  with  9  c.c.  of  medium;  i  c.c.  of  tube 
No.  2  to  tube  No.  3  (with  9  c.c.  of  medium),  following  the  other  directions 
as  already  given.  Plate  out  as  already  explained,  and  watch  developments. 
In  Petri  dish  No.  i  the  number  of  bacterial  growths  (colonies)  will  no  doubt 


FIG.  36. — Petri  dish. 


These    dishes    are  among  the    essentials  in  the  bacteriological 
laboratory.     (Williams.) 


be  so  great  as  to  make  counting  impossible.  Petri  dish  No.  2  may  contain 
360  colonies,  and  dish  No.  3  may  contain  not  more  than  40.  An  average 
is  obtained  by  repeating  the  test  (using  the  same  milk  sample)  a  number 
of  times.  In  the  above  milk  sample  the  average  may  be  42,000  microbes  per 
c.c.  If  the  bacterial  content  is  high,  it  is  necessary  to  extend  the  attenuation 
four  and  even  five  times. 

If  it  is  desired  to  determine  the  number  of  bacteria  per  gram  of  dry  soil, 
it- will  be  necessary  to  carefully  weigh  a  small  quantity  (i  gm.,  more  or  less) 
of  average  soil,  triturate  the  entire  sample  with  say,  100  c.c.  of  sterile  distilled 
water,  and  from  this  make  the  dilution  cultures  as  above  described,  using 
i  c.c.  or  less  of  the  soil  triturate.  To  compute  the  number  of  bacteria  per 
gram  of  dry  soil,  it  will  now  be  necessary  to  determine  the  moisture  percentage 
in  a  sample  of  soil  taken  from  the  same  place  as  the  sample  which  was  used 
in  making  the  triturate.  The  solution  is  simple.  We  will  suppose  the  tritu- 
rate sample  weighed  0.856  gm.  and  the  number  of  bacteria  found  was 


6o 


PHARMACEUTICAL   BACTERIOLOGY. 


3,000,000;  and  the  percentage  of  moisture  was  10.  From  these  data  it  would 
be  found  that  i  gm.  of  dry  soil  will  contain  3,855,011  microbes. 

The  above  is  sufficient  to  make  clear  how  one  might  proceed  to  deter- 
mine the  number  of  microbes  in  and  upon  old  pills,  tablets,  powders;  on  one 
ivory  vaccine  tip,  in  one  glycerinated  vaccine  tube,  in  i  c.c.  of  bacterial 
vaccine,  antitoxin,  syrup,  tincture,  fluidextract,  camphor  water,  distilled 
water,  sewage,  drinking  water,  etc.  Naturally,  great  caution  and  care  must 
be  observed  to  avoid  errors  and  faulty  conclusions.  In  fact,  no  one  should 
attempt  such  work  in  actual  practice  until  after  considerable  preliminary 
laboratory  experience. 

It  is  not  practicable  nor  is  it  necessary  to  give  fuller  information  regarding 
bacterial  cultures.  We  have  not  touched  upon  the  various  methods  for 
determining  whether  or  not  the  microbes  under  investigation  are  essentially 


FIG.  37. — Graduated  fermentation  tube.     These  tubes  are  required  for  gas  determination 
with  colon  bacillus  and  other  gas-forming  micro-organisms. 

aerobic  or  essentially  anaerobic;  the  manner  of  determining  the  thermal  death- 
point;  relationship  of  rate  of  growth  to  temperature,  etc.  We  have  said 
nothing  of  the  use  of  indicators  added  to  culture  media,  as  litmus,  rosolic 
acid,  and  phenolphthalein,  nor  have  we  explained  the  special  use  of  special 
culture  media  in  determining  the  nature  and  identity  of  bacteria.  These 
and  many  other  details  we  must  omit,  merely  stating  that,  should  it  become 
desirable  to  make  such  investigations,  the  necessary  information  must  be 
secured  elsewhere,  as  in  some  standard  laboratory  guide  in  bacteriological 
technic. 

The  following  outline  of  special  methods  will  serve  as  a  guide  in  making 
bacteriological  examinations  of  soils,  air,  pharmaceuticals,  liquids,  etc. 

D.  Cultur ing  Soil  Bacteria. — Soil  is  a  mixture  of  dead  and  decayed  organic 
matter,  sand  and  living  organisms  and  their  spores.  Near  the  surface  the 
soil  contains  large  numbers  of  bacteria,  from  10,000  to  10,000,000  per  gram, 


BACTERIOLOGICAL   TECHNIC.  6 1 

and  more.  In  fact  the  fertility  of  the  soil  is  practically  proportional  to  the 
number  of  bacteria  present.  Most  species  of  soil  bacteria  are  harmless  to 
man  though  the  bacilli  of  tetanus  (lockjaw),  of  typhoid  fever,  of  malignant 
edema,  of  anthrax,  and  of  pus  formation  may  be  present.  The  tetanus 
germ  is  quite  common  in  garden  soils  and  the  anthrax  germ  is  apt  to  occur 
in  cattle  pens,  pastures  and  other  places  frequented  by  cattle.  Other  soil 
bacteria  are  decidedly  useful  as  will  be  more  fully  explained  elsewhere. 

Some  soil  bacteria  (the  nitrifiers)  do  not  grow  on  the  usual  media  while 
others  thrive  exceedingly  well  in  such  media.  Anaerobic  forms  must  be 
cultured  in  the  absence  of  air  or  oxygen. 

The  root  nodule  bacteria  of  the  leguminosae  can  be  grown  readily  on 
gelatin  or  agar.  The  tubercles  or  nodules  must  be  thoroughly  cleansed  and 
repeatedly  washed  in  boiled  distilled  water,  then  rinsed  for  ten  seconds  in  a 
i-iooo  corrosive  sublimate  solution,  and  finally  thoroughly  rinsed  (three 
minutes)  in  boiled  distilled  water.  Crush  several  of  the  sterilized  nodules 
in  a  sterile  watch  crystal,  by  means  of  a  sterile  glass  rod  and  from  this  make 
the  dilution  plate  cultures  and  set  aside  at  room  temperature.  Colonies  of 
small  motile  bacteria  (Rhizobium  mutabile)  will  appear  in  about  four  days. 

To  test  the  soil  bacterially,  select  thoroughly  mixed  samples  and  plate  out 
as  already  suggested,  using  every  precaution  to  prevent  the  introduction  of 
extraneous  germs.  Cultures  can  also  be  made  from  internal  plant  tissues 
by  following,  in  general,  the  directions  given  under  root  nodule  bacteria,' 
excepting  that  after  the  washing  and  rinsing,  the  root,  instead  of  being 
crushed,  is  cut  or  broken  across  and  the  inoculation  material  is  taken  from 
the  inner  tissue  by  means  of  a  platinum  needle  or  scalpel. 

E.  Bacteria  of  the  Air. — Air  currents  carry  the  germ-laden  dust  and  dirt 
particles.  The  number  and  kind  of  air  bacteria  depends  upon  environment, 
climatic  conditions,  moisture,  sunlight,  etc.  The  air  currents  are  the  main 
factors  in  germ  dissemination.  Spores  and  dry  (though  not  dead)  bacilli 
may  be  carried  many  miles.  Air  microbes  are  derived  from  the  soil  surface 
and  from  the  objects  surrounded  by  the  air.  Bacteria  are  exhaled  with  the 
breath  and  are  carried  and  distributed  from  and  by  animals,  plants  and 
clothing.  The  air  carries  pus  germs,  tubercle  bacilli,  anthrax  bacilli  and 
their  spores,  besides  other  pathogenic  microorganisms,  including  also  yeast 
cells  and  the  spores  of  higher  fungi. 

Air  microbes  may  be  studied  by  exposing  a  Petri  dish  containing  steril- 
ized agar  or  gelatin,  for  two  minutes  or  longer.  The  number  of  colonies 
that  will  appear  will  depend  upon  the  locality,  season,  air  moisture,  etc. 
To  determine  the  number  of  microbes  in  a  given  volume  of  air  the  Sedgwick- 
Tucker  aerobioscope  is  used,  though  similarly  constructed  apparatus  may 
be  made  by  any  fairly  skillful  student.  The  aerobioscope  consists  of  a  glass 
cylinder  as  shown  in  the  illustration.  The  open  ends  are  plugged  with  cotton. 


62  PHARMACEUTICAL   BACTERIOLOGY. 

Granulated  sugar  is  loosely  packed  into  the  narrow  end  and  all  is  then 
sterilized  in  a  hot-air  sterilizer  (not  over  120°  C.).  Pass  a  given  quantity  of 
air  through  the  aerobioscope  by  attaching  an  aspirator  bottle  to  the  narrow 
end  and  allowing  a  given  volume  of  water  to  run  out  of  the  bottle.  The 
volume  of  air  drawn  through  equals  the  volume  of  water  run  from  the  bottle. 
Of  course  the  cotton  plug  is  removed  from  the  larger  end  of  tube  while  the 
water  is  running.  The  bacilli  and  spores  are  caught  in  the  sugar,  while  the 
air  passes  through.  Replace  cotton  plug  and  shake  the  sugar  into  the 
larger  end  of  tube.  Remove  cotton  plug  again  and  pour  in  about  10  to  15 
c.c.  of  liquefied  (40°  C.,  not  hot)  gelatin.  Roll  the  tube  held  horizontally. 
The  gelatin  dissolves  the  sugar  and  mixes  with  it.  Roll  on  ice  to  hasten  the 
hardening  of  the  gelatin.  Set  aside  in  incubator,  at  room  temperature 
(20°  C.,  about).  The  number  of  colonies  which  appear  indicates  approx- 


FIG.  38. — Aerobioscope    after     Sedgwick-Tucker,    plugged    with    cotton.      The     larger 
end  in  which  the  cultureing  is  done  is  ruled  to  facilitate  the  counting  of  colonies. 

imately  the  number  of  microbes  in  the  volume  of  air  aspirated.  Let  us 
suppose  that  the  number  of  colonies  was  125,  the  volume  of  air  aspirated  10 
liters,  from  which  we  would  get  1250  bacteria  per  cubic  meter  of  air. 

F.  Bacteria  of  Liquid  Substances. — The  bacteria  of  water,  milk,  tinctures, 
fluidextracts,  aquae,  aerated  waters,  mineral  waters,  distilled  water,  broth, 
and  liquids  generally,  can  be  studied  quantitatively  in  a  comparatively  simple 
manner.  By  means  of  a  sterile  i  c.c.  graduated  pipette,  run  o.i  c.c.  to 
0.5  c.c.  of  the  liquid  into  the  center  of  a  sterilized  petri  dish,  pour  upon  this 
enough  (about  10  c.c.)  melted  (sterile)  agar  or  gelatin  and  mix  by  tilting 
the  dish  slightly  from  side  to  side.  Set  aside  for  the  medium  to  harden 
and  incubate  at  the  room  temperature,  or  at  25°  C.,  if  quicker  results  are 
desired.  This  method  is  satisfactory  if  the  number  of  bacilli  present  is 
comparatively  small.  If  very  abundant,  dilutions  must  be  made  in  the 
manner  already  described. 

The  following  general  suggestions  should  be  observed  in  making  bacterio- 
logical determinations  of  liquids: 

a.  Containers  for  samples  (other  than  the  original  containers)  must  be 
sterile  and  closed  with  sterile  corks  or  cotton  plugs.  If  the  samples  are  to 
be  carried  any  distance  they  should  be  packed  in  ice.  In  no  case  is  it  wise  to 
keep  a  sample  longer  than  forty-eight  hours  before  culturing  it.  If  the 
sample  is  to  be  examined  within  two  or  three  hours  after  collecting  it, 
placing  on  ice  is  not  absolutely  necessary. 


BACTERIOLOGICAL   TECHNIC.  63 

b.  Every  sample  should  be  thoroughly  mixed  before  making  cultures. 
Shake  well,  about  twenty  times.     This  is  very  important. 

c.  All  glassware,  pipettes,  etc.,  must  be  thoroughly  sterilized  by  washing, 
use  of  disinfectants,  rinsing,  wiping,  hot  air  and  steam  sterilization,  etr. 

d.  Incubate  at  room  temperature,  as  a  rule.     Colonies  will  begin  to 
appear  in  forty-eight  hours.     The  maximum  development  will  be  in  three  or 
four  days,  in  most  instances,  provided  the  temperature  does  not  fall  below 
20°  C. 

e.  As  a   rule  the    presence  of  abundant  gelatin-liquefying  organisms 
may  be  looked  upon  with  suspicion.     Certain  sewage  organisms  liquefy 
gelatin  very  actively. 

f.  The  colon  bacillus  and  some  sewage  cocci  give  pink  colonies  with 
lactose  litmus  agar  medium.     The  cocci  colonies  are  a  deeper  vermilion 
than  the  colon  colonies'.     Sewage-contaminated  water  will  show  many  pink 
colonies. 

g.  Certified  milk  (just  delivered)  should  not  show  more  than  from  1000 
to  10,000  colonies  per  c.c. 

h.  Wholesome  uncertified  milk  should  not  show  more  than  from  30,000 
to  50,000  colonies  per  c.c.  The  number  of  colonies  permissible  varies  in 
different  states  and  in  different  localities  in  the  same  state.  The  number  of 
colonies  may  range  from  25,000  to  1,000,000  per  c.c.,  and  even  more,  and  yet 
the  milk  may  be  pronounced  wholesome.  No  pink  colonies  should  be  present. 
No  pus  cells  should  be  present  (centrifugalized  sediment). 

i.  Good  drinking  water  should  not  show  more  than  50  to  100  colonies 
per  c.c.  and  there  should  be  no  pink  colonies,  only  a  few  liquefying  colonies 
(i-io)  and  most  of  the  colonies  should  develop  best  at  20°  C.  If  50  per 
cent,  of  the  colonies  develop  best  at  30°  to  38°  C.  this  indicates  probable 
sewage  contamination  or  contamination  with  intestinal  bacteria.  This 
differential  temperature  test  is  considered  of  importance  in  the  bacterial 
examination  of  drinking  waters.  Normal  water  gives  a  proportion  of  i 
colony  of  high  temperature  organisms  to  from  25  to  50  colonies  of  low 
temperature  organisms.  In  sewage  contaminated  water  the  proportion  is  i 
to  4  and  even  less. 

j.  Thus  far  there  are  no  standards  for  the  bacteriological  testing  of  phar- 
•maceuticals.  Tinctures  and  fluidextracts  should  show  only  few  colonies  per 
c.c.,  not  over  30  to  60.  Sera  should  show  none.  Well  prepared  and  prop- 
erly ripened  small-pox  vaccine  should  show  only  a  few  colonies  per  point  or 
per  glycerinated  tube.  Aquae  often  show  abundant  colonies,  from  10,000 
to  10,000,000  per  c.c.  and  more. 

k.  The  colon  bacillus  should  not  be  present  in  drinking  water,  in  milk 
or  in  pharmaceuticals.  If  present,  it  indicates  sewage  or  other  objectionable 
contamination.  The  colon  bacillus  is  motile  in  young  broth  cultures,  forms 


64  PHARMACEUTICAL   BACTERIOLOGY. 

no  spores,  is  gas-  (dextrose  broth  cultures  in  fermentation  tube)  and  indol- 
forming,  reduces  nitrates  to  nitrites,  does  not  liquefy  gelatin  and  is  not 
stained  by  Gram's  method. 

1.  Syrups  of  all  kinds,  unless  very  carefully  prepared  and  carefully  kept 
to  prevent  fermentation,  are  apt  to  show  numerous  bacteria,  yeasts  and 
moulds.  Any  syrup  showing  signs  of  yeast  fermentation  (gas  bubbles, 
vinous  odor)  or  mouldiness,  is  not  fit  for  use  and  should  be  rejected.  The 
attempt  to  render  it  usable  by  boiling  is  unsatisfactory,  furthermore  the 
changes  produced  by  the  organisms  are  always  objectionable  and  cannot  be 
rectified  by  heating  or  by  other  methods  of  sterilization. 

m.  Recent  investigations  have  shown  that  many  of  the  marketed  (bottled) 
mineral  waters  contain  numerous  bacteria,  from  10,000  to  300,000,000  and 
more  per  c.c.  In  some  cases  colon  bacilli  have  been  found.  These  find- 
ings prove  that  in  many  instances  the  methods  of  bottling  must  be  careless 
or  otherwise  unsatisfactory  since  sewage  contamination  is  not  reasonably 
possible  under  proper  sanitary  conditions.  Undoubtedly  the  contamination 
is  in  some  instances  due  to  reused  and  inadequately  cleaned  and  sterilized 
containers  and  in  other  instances  to  impure  and  inadequately  sterilized 
mineral  water.  A  popular  opinion  prevails  that  the  chemicals  in  the  min- 
eral waters  are  sufficiently  germicidal  to  destroy  bacteria  but  this  is  not  the 
case.  Bacteria  may  develop  actively  in  a  great  variety  of  solutions  of  high 
concentration  provided  such  solutions  are  chemically  balanced.  Loeb, 
Osterhaut  and  others  have  shown,  for  example,  that  ocean  water  is  chemically 
balanced,  thus  being  suitable  to  maintain  life  in  a  great  variety  of  organisms. 

G.  Bacteria  in  Canned  Fruits. — The  work  recently  demanded  by  the  pure 
food  laws  (federal  and  state)  has  shown  that  such  food  substances  as  canned 
fruits  of  all  kinds,  including  jams,  jellies,  preserves,  catsups,  tomato  pastes, 
etc.,  are  frequently  highly  contaminated  with  yeast  cells,  moulds  and  their 
spores,  and  other  higher  fungi,  and  bacteria.  It  is,  however,  evident  that 
the  food  products  named  may  be  quite  free  from  such  contamination  as 
may  be  seen  from  the  examination  of  canned  food  products  prepared  by 
the  careful  housewife.  That  manufacturers  may  approximate  the  home 
condition  is  demonstrated  by  the  fact  that  factory  products  are  found 
on  the  market,  which  are  quite  free  from  contamination. 

Since  wholesome  ripe  fruit  contains  yeast  cells,  bacteria  and  mould  in 
very  small  numbers  only,  and  since  most  of  these  organisms  are  removed  in 
the  various  steps  of  the  processing,  as  washing,  peeling,  steaming,  etc.,  it  is 
evident  that  the  finished  factory  product  should,  like  the  home-made 
product,  contain  these  organisms  in  negligibly  small  numbers  only,  provided, 
of  course,  that  wholesome  fruit  is  used.  However,  most  of  the  factory  samples 
thus  far  examined  have  shown  numerous  dead  yeast  cells,  mould  spores, 
mould  hyphae,  and  bacteria,  indicating  the  use  of  fruit,  fruit  pulp,  fruit 


BACTERIOLOGICAL   TECHNIC.  65 

juices,  fruit  refuse,  etc.,  which  was  decomposed  or  undergoing  fermentation 
or  decomposition  prior  to  or  at  the  time  of  manufacture.  The  organisms 
named  prevail  in  varying  amounts  in  different  products.  Yeast-organisms 
are  apt  to  predominate  in  jellies,  fruit  juices  and  fruit  pulp;  bacteria  in 
catsups  and  pastes;  and  moulds  in  certain  fruits  as  strawberries,  blackberries 
and  raspberries. 

The  presence  of  numerous  dead  yeast  cells  (1,000,000  to  50,000,000  per 
c.c.)  is  evidence  that  the  material  was  undergoing  alcoholic  fermentation 
just  prior  to  or  at  the  time  of  manufacture.  Tomato  pastes  have  been  found 
on  the  market  showing  over  400,000,000  bacteria  per  c.c.  besides  numerous 
yeast  cells  and  considerable  mould.  The  bacterial  content  of  catsups  is 
apt  to  run  high,  from  10,000,000,  to  50,000,000  and  more  per  c.c.  Not 


m 


Areas 

I  sa.  mm.. 


Cu.bic  Contents 

So*,SW:> 

O.Z.C.mm^. 
O.ooSC.nim.. 


FIG.  39. — Counting  apparatus  for  mould,  yeast  cells  and  spores.  From  the  measuring 
values  marked  on  the  slide  it  is  easy  to  determine  the  number  of  mould  hyphal  clusters, 
yeast  cells  and  spores  per  c.c.  of  the  substance  under  examination.  Used  with  No.  2 
ocular  and  No.  3  and  No.  5  objectives. 

The  rulings  are  as  follows:  There  are  75  square  millimeters  in  the  entire  area,  of 
three  squares  of  25  square  millimeters  each.  The  one-millimeter  areas  are  to  be  used  in 
determining  the  quantity  of  mould,  dirt,  sand  and  other  impurities  present.  The  one- 
millimeter  areas  indicated  black  in  the  figure  are  marked  off  into  1/25  (0.04)  square 
millimeters.  These  smallest  areas  are  used  in  making  spore  and  yeast  cell  counts.  The 
depth  is  o. 2  millimeter. 

We  will  suppose  that  a  given  fruit  sample,  as  strawberry  jam,  shows  30  yeast  cells 
in  the  smallest  area  (0.2  mm.  Xo.o4  mm.  =0.008  cm.),  then  i  c.c.  of  the  substance  would 
contain  3,750,000  yeast  cells. 

including  the  vinegar  bacteria,  which  are  introduced  into  catsups  and  pastes, 
such  high  bacterial  content  is  generally  due  to  bacterial  development  during 
or  after  manufacture.  The  presence  of  mould  organisms  and  their  spores 
(other  than  penicillium)  indicates  the  use  of  mould-infested  fruit.  Pen- 
icillium,  which  is  entirely  saprophytic  in  habit,  may  develop  after  manu- 
facture, particularly  on  the  surface  of  inadequately  sterilized  fruit  products 
in  containers  not  entirely  filled. 

"Swelling"  of  cans  containing  fruit  products  is  generally  due  to  yeast 
development  though  it  may  also  be  due  to  bacterial  activity,  and  indicates 
inadequate  sterilization  of  either  the  container  or  of  the  fruit  or  both.  Exam- 
ination will  show  the  presence  of  living  yeast  cells,  or  bacteria,  perhaps  air 
bubbles,  and  the  characteristic  vinous  odor  of  yeast  may  be  noted. 
5 


66  PHARMACEUTICAL   BACTERIOLOGY. 

Based  upon  such  conditions  as  can  be  made  to  prevail  in  carefully  oper- 
ated factories,  the  following  may  be  given  as  the  limits  of  the  number  of 
organisms  permissible  in  the  fruit  products  under  discussion. 

a.  Yeast  cells,  either  living  or  dead,  not  to  exceed  1,000,000  per  c.c. 

b.  Mould  spores  not  to  exceed  50,000  per  c.c. 

c.  Hyphal  clusters  and  hyphal  fragments  not  to  exceed  10,000  per  c.c.; 
or  not  over  50  per  cent,  of  separate  and  distinct  fields  of  view  under  the 
compound  microscope  should  show  hyphal  clusters  or  hyphal  fragments. 

d.%  Bacteria  (either  living  or  dead  but  not  including  vinegar  bacteria  in 
products  to  which  vinegar  is  added)  not  to  exceed  5,000,000  per  c.c. 

The,  above  figures  apply  only  to  fruit  products  supposedly  made  from 
comparatively  fresh  fruits  and  fresh  fruit  juices.  The  yeast,  bacterial  and 
spore  counts  are  made  with  a  Thoma-Zeiss  hemacytometer  (Turck  ruling) 
using  a  No.  5  (1/5  in.)  objective  with  No.  2  (i  in.)  ocular. 

H.  Quantitative  and  Qualitative  Bacteriological  Testing. — The  following 
will  serve  as  a  general  outline  of  bacteriological  analyses  which  may  be  made 
in  food  and  drug  laboratories.  The  substances  which  require  such  bacterio- 
logical examination  include  catsups,  tomato  pastes,  vinegars,  water  supplies, 
mineral  waters,  milk,  ice  creams,  any  and  all  substances  which  are  suspected 
to  be  sewage  contaminated,  etc.,  etc. 

The  sequence  of  processes  here  given  bear  a  progressive  relationship. 
Whether  process  II  is  carried  out  will  depend  upon  the  findings  under  I  and 
whether  III  shall  be  undertaken  will  depend  upon  the  findings  under  II.  The 
essential  facts  to  be  ascertained  are  whether  or  not  there  is  possible  sewage 
contamination  as  indicated  by  the  presence  of  the  colon  bacillus,  sewage 
streptococci  and  possibly  the  typhoid  bacillus.  The  typhoid  agglutinating 
tests  are  apt  to  prove  unsatisfactory.  In  most  instances  this  test  will  be 
unnecessary  as  the  presence  of  the  colon  bacillus  is  evidence  that  the 
food,  drug  or  drink  is  contaminated  with  sewage  and  is  hence  unfit  for 
human  use. 

I.  Direct  Count. — For  this  purpose  the  Thoma-Zeiss  hemacytometer 
with  Turck  ruling1  is  used  (No.  2  ocular  with  1/5  in.  objective)  which  can  be 
secured  from  any  bacteriological  supply  house.  The  instructions  for  using 
it  can  be  obtained  from  the  dealer,  though  the  measuring  values  indicated 
on  the  hemacytometer  are  sufficient  to  indicate  the  manner  of  making  the 
counts.  The  rulings  generally  used  for  bacterial  countings  are  1/25  sq.  mm. 
X  i/io  mm.  deep,  making  an  area  of  1/250  cu.  mm.,  or  reduced  to  decimal 
fractions,  0.04  sq.  mm.  X  o.i  mm.  deep  =  0.004  cu.  mm.  We  will  suppose 
that  the  average  of  20  counts  shows  5  bacilli,  then  i  cu.  mm.  would  contain 
1,250  bacilli  or  1,250,000  in  i  c.c. 

The  direct  count  is,  in  many  instances,  very  unsatisfactory  for  several 

1  To  render  the  ruled  lines  visible  rub  a  very  soft  pencil  over  the  ruled  area. 


BACTERIOLOGICAL   TECHNIC.  67 

reasons.  Particles  other  than  micro-organisms  may  be  mistaken  for  bacilli 
or  cocci  and,  furthermore,  it  cannot  be  known  for  a  certainty  that  the  organ- 
isms are  dead  or  alive.  If  they  are  present  in  great  abundance  (10,000,000  to 
100,000,000  and  more  per  c.c.),  ordinary  smear  preparations  maybe  stained, 
using  methyl  blue  or  fuchsin.  Dead  bacilli,  that  is  those  which  have  been 
dead  for  some  time,  do  not  take  the  stain  well,  due"  to  the  fact  that  the  cell- 
plasm  is  disintegrated. 

Tomato  pastes,  anchovy  pastes,  catsups,  some  mineral  waters  and 
similar  preparations,  may  contain  bacteria  in  such  numbers  that  dilutions 
are  desirable  or  necessary  to  make  counting  possible.  A  dilution  of  one  in 
ten  will,  as  a  rule,  be  sufficient.  Weigh  or  measure  one  part  (i  gm  or  r 
c.c.)  of  the  substance,  add  it  to  nine  parts  filtered  distilled  water  and  mix 
thoroughly  by  shaking. 

If  the  direct  count  shows  bacilli  in  great  numbers  or  if  for  any  reason 
sewage  contamination  is  suspected,  and  also  to  determine  the  number  of 
living  bacilli  and  spores  suspected,  proceed  as  follows: 

II.  Plate  Culture  Counts. — Make  one  set  of  plate  cultures,  using  lactose 
litmus  agar,1  and  incubate  at  20°  C.  Make  a  second  set  of  plate  cultures, 
also  upon  lactose  litmus  agar,  and  incubate  at  38°  C.  The  usual  dilution 
methods  are  followed  when  necessary,  using  preferably  o.i  c.c.  quantities  for 
the  plates.  This  temperature  differential  test  is  considered  of  great  impor- 
tance. Colon  bacilli  and  other  micro-organisms,  whose  natural  habitat  is 
the  intestinal  canal,  will  develop  actively  at  the  higher  temperature  (38°  C.), 
whereas  the  usual  air,  soil  and  water  bacteria  develop  best  at  the  lower  tem- 
perature (20°  C.).  If  the  high  temperature  colonies  approximate  the  low 
temperature  colonies,  sewage  contamination  may  be  suspected.  If  in  addi- 
tion many  of  the  high  temperature  lactose  litmus  agar  colonies  show  pink  or 
light  vermilion,  the  sewage  contamination  is  practically  proven.  The  colon 
bacillus,  as  well  as  sewage  streptococci,  give  pink  colonies,  the  latter  being  the 
brighter,  more  vermilion  in  coloration,  due  to  the  formation  of  acid  (in  the 
fermenting  lactose) .  Examine  the  pink  colonies  under  the  microscope.  The 
colon  microbe  is  rod-shaped,  rather  thick,  non-sporing.  and  shows  motility  in 
recent  broth  cultures,  whereas  the  streptococci  are  smaller  and  are  not  rod- 
shaped.  High  temperature  colonies  as  compared  with  low  temperature  col- 
onies should  not  exceed  1:100  or  1:25.  If  the  proportion  is  1:4  or  less, 
sewage  contamination  is  very  likely.  After  36  hours  the  pink  colonies  may 
turn  blue,  due  to  the  development  of  ammonia  and  amines. 

Naturally  the  high  temperature  colonies  must  be  studied  within  twenty- 
four  to  thirty  hours  whereas  the  low  temperature  cultures  require  much  more 
time,  two  to  four  days. 

1  Add  i  per  cent,  of  lactose  to  the  usual  agar  medium  and  enough  tincture  of  litmus- 
to  give  it  a  lilac  tinge. 


68  PHARMACEUTICAL   BACTERIOLOGY. 

If  the  temperature  and  color  differential  tests  indicate  sewage  contamina- 
tion, then  the  following  additional  tests  should  be  carried  out. 

III.  Indol  Reaction  and  Gas  Formula. — The  indol  reaction  has  already  been 
explained.  The  gas  formula  is  determined  as  follows:  To  sets  of  four  grad- 
uated fermentation  tubes  containing  glucose  bouillon  and  lactose  bouillon, 
add  o.i,  0.2,  0.5,  and  10  c.c.  of  the  suspected  liquid.  If  gas  formation 
is  observed  the  presence  of  colon  bacilli  may  be  suspected.  If  the  o.i  c.c. 
tubes  show  gas  formation  then  the  presence  of  colon  bacilli  may  be  assumed. 
Fill  the  bulb  of  a  tube,  showing  gas  formation,  with  a  2-per  cent,  solution  of 
sodic  hydrate,  hold  thumb  tightly  over  the  opening  and  mix  contents  by 
tilting  back  and  forth  carefully.  The  portion  of  gas  absorbed  is  CO2 
whereas  the  unabsorbed  portion  is  supposedly  hydrogen.  The  colon  bacillus 
shows  a  gas  formation  of  1/3  hydrogen.  Of  course  the  total  volume  of  gas  is 
recorded  before  the  sodic  hvdrate  is  added. 


FIG.  40. — Colon  bacillus.  This  microbe  is  quite  large,  in  the  comparative  sense,  and 
is  morphologically  typical  of  the  group  bacillus.  The  flagellae  are  few  in  number  and 
comparatively  long. 

The  gas  formula  with  a  positive  indol  reaction  is  practically  conclusive 
as  far  as  the  presence  of  the  colon  bacillus  is  concerned.  Add  to  this  the 
other  tests  and  we  have  presumptive  evidence  of  sewage  contamination,  and 
any  article  of  food  or  drink  showing  such  contamination  is  unfit  for  human 
consumption. 

The  colon  bacillus,  ,the  bacilli  of  the  hog  cholera  group  and  others 
have  the  power  of  reducing  neutral  red;  producing  a  greenish-yellow 
fluorescence.  For  this  reaction  use  glucose  bouillon  to  which  has  been  added 
i  per  cent,  of  a  0.5  per  cent,  solution  of  neutral  red.  In  examining  milk,  the 
pus  cell  and  leucocyte  count  is  considered  important;  centrifugalize  10  c.c. 
of  milk  for  thirty  minutes,  pour  off  supernatant  milk  and  mix  residue  with 


BACTERIOLOGICAL   TECHNIC.  69 

0.5  c.c.  normal  salt  solution  and  make  counts  of  pus  cells  and  leucocytes 
per  c.c.  from  the  amount  (0.5  c.c.).  Abundant  pus  cells  and  leucocytes  indi- 
cate abscess  or  other  pathological  condition  of  milk  ducts  or  glands.  This 
test  is,  however,  of  little  significance  excepting  in  the  hands  of  authorities 
on  diseases  of  cows.  It  is  stated  that  as  many  as  100,000  leucocytes  per  c.c. 
may  occur  in  apparently  healthy  animals. 

Gelatin-liquefying  organisms  may  be  looked  upon  with  suspicion 
when  found  in  milk,  water  and  other  liquid-food  substances  intended  for 
human  consumption,  as  has  already  been  explained. 

It  should  be  borne  in  mind  that  the  colon  bacillus  is  one  of  a  group  of 
some  fifteen  or  more  species  and  varieties  of  closely  related  micro-organisms 
which  resemble  each  other  in  the  following  particulars: 

1.  Do  not  form  spores. 

2.  Do  not  liquefy  gelatin. 

3.  Produce  acid  in  milk  and  cause  milk  coagulation. 

4.  Produce  acid  and  gas  in  glucose  and  lactose  media. 

5.  Produce  acid  and  gas  in  bile-salt-glucose  broth. 

6.  Grow  well  in  temperatures  ranging  from  38°  to  42°  C. 

In  differentiating  the  colon  bacillus,  remember  that  this  organism  is 
rod-shaped  (2  to  3/1  long  by  0.5  to  o.6/*  wide),  is  motile,  produces  indol, 
gives  rise  to  pink  colonies  on  lactose  (or  glucose)  litmus  agar  and  reduces 
neutral  red  glucose  (or  lactose)  agar  with  a  greenish-yellow  fluorescence. 

It  should  also  be  remembered  that  sewage  is  a  highly  complex  substance 
and  contains  micro-organisms  in  great  variety  and  in  great  abundance. 
Among  the  organisms  present  are  species  of  Spirillum,  Vibrio,  Proteus  and 
Beggiatoa  in  addition  to  the  bacilli  and  streptococci  already  mentioned. 
The  typhoid  bacillus  does  not  thrive  well  in  sewage.  The  number  of  bac- 
teria present  in  crude  or  ordinary  sewage  (domestic,  city,  hospital,  mixed, 
etc.)  ranges  from  1,000,000  to  100,000,000  and  more  per  c.c.  The  work 
of  these  organisms  is  to  break  down  and  render  soluble  and  assimilable 
(for  plants)  the  organic  matter  composing  the  sewage,  thus  assisting  the  work 
of  rotting  bacteria  generally. 

The  following  is  a  tabulation  of  the  bacteriological  testing  that  should 
be  made  of  foods  (including  pastes,  catsups,  milk,  ice  creams,  water  supplies, 
mineral  waters,  alcoholic  beverages,  etc.)  that  may  show  an  excess  of  bacterial 
growth  or  which  may  be  sewage  contaminated: 

BACTERIOLOGICAL  EXAMINATION. 

I.  Direct  Count. 

1.  Bacilli  per  c.c 

2.  Cocci,  per  c.c . 


70  PHARMACEUTICAL   BACTERIOLOGY. 

II.  Plate  and  Tube  Cultures.     (Lactose-litmus-agar.) 

1.  Temperature  differential  test. 

a.  (20°  C.)   Colonies  per  c.c 

b.  (38°  C.)   Colonies  per  c.c 

2.  Color  differential  test. 

a.  Pink  and  yellow  colonies  per  c.c 

c.  Not  pink  or  yellow  colonies  per  c.c.     . 

3.  Colorless  gelatin  liquefying  colonies  per  c.c. 

4.  Neutral  red  reduction,  +  or  — . 

5.  Indol  reaction,  +  or  — . 

6.  Gram  stain  behavior,  +  or  — . 

7.  Gas  (hydrogen)  formula. 

III.  Agglutinating  tests  for  Typhoid  Germs. 

*  8.  Staining  Bacteria. 

Staining  consists  of  the  infiltration  of  the  cell-substance  with  solutions  of 
various  coloring  materials  obtained  for  the  most  part  from  the  group  of  coal- 
tar  derivatives  known  as  the  aniline  dyes.  As  is  generally  known,  different 
cells  and  different  portions  of  one  and  the  same  cell  react  differently  with 
the  various  dyes  used.  This  peculiar  behavior  brings  out  contrasts  in 
appearances  which  aid  very  materially  in  determining  the  morphological 
characters.  The  prime  object,  therefore,  in  using  stains  is  to  aid  in  the  study 
of  cell  morphology.  Different  bacteria  react  differently  with  the  several 
stains  used.  Some  species  take  certain  stains  very  readily,  while  they  are 
quite  indifferent  to  other  stains.  The  vegetative  cell  stains  much  more 
readily  than  do  the  spores.  In  fact,  spores  are  stained  with  great  difficulty; 
however,  after  they  are  once  thoroughly  stained  they  hold  the  color 
persistently. 

The  dyes  which  may  be  used  in  bacteriologic  work  are  of  many  kinds, 
differing  as  to  color  and  as  to  staining  powers  with  different  cells,  cell-con- 
tents, and  cell-parts.  They  are  usually  classified  as  acid  or  basic.  Eosin, 
acid  fuchsin,  and  picric  acid  are  acid  stains,  and  are  said  to  be  diffuse  in 
their  effects,  having  no  special  affinity  for  any  special  cell  structure.  Fuch- 
sin, methylene  blue,  and  gentian  violet  are  basic,  and  appear  to  have  special 
attraction  for  bacteria  and  for  plasmic  and  nuclear  substances  of  cells  gener- 
ally, for  which  reasons  they  are  most  generally  employed  as  bacterial  stains. 
Fuchsin  is,  in  fact,  about  the  only  efficient  stain  for  endospores,  while 
gentian  violet  and  methylene  blue  are  excellent  stains  for  the  bacterial  cell- 
wall. 

It  is  known  that  certain  substances  possess  the  property  of  preparing  the 
bacterial  cells  in  such  a  way  as  to  induce  them  to  take  up  the  dye  more 


BACTERIOLOGICAL   TECHNIC.  7 1 

readily,  thus  intensifying  the  stain,  as  aniline  oil  and  carbolic  acid.  Such 
substances  are  called  mordants,  and  may  be  used  separately  or  added  directly 
to  the  stain  itself. 

Certain  liquids  or  solutions  remove  the  stain  from  the  bacterial  cell 
more  or  less  readily,  as  water  and  alcohol,  but  more  particularly  solutions  of 
acids.  Such  substances  are  quite  generally  employed  for  removing  any 
excess  of  stain  from  the  bacterial  cell  or  from  the  matrix  in  which  the  bacteria 
are  fixed  or  embedded.  Acidulated  (with  HC1)  alcohol  is  most  commonly 
employed.  Ordinarily,  rinsing  in  a  small  stream  of  water  is  sufficient. 
Some  bacteria  resist  the  decolorizing  process  with  acids  more  strongly  than 
others,  and  are  said  to  be  acid  fast  or  acid  proof,  as,  for  example,  the  bacilli 
of  leprosy  and  of  tuberculosis,  while  the  great  majority  of  species  give  up 
the  stain  very  readily.  It  is  a  fact  that  one  and  the  same  species  of  microbe 
reacts  variably  with  one  and  the  same  stain,  depending  upon  a  variety  of 
causes.  Moderate  heat  hastens  and  intensifies  the  staining. 

For  ordinary  purposes  a  single  stain  only  is  used,  but  sometimes  struc- 
tural differences  are  more  clearly  shown  by  what  is  known  as  double  or  con- 
trast staining.  Take,  for  example,  a  spore-bearing  microbe,  as  that  of 
anthrax.  The  spores  may  be  stained  by  means  of  carbol  fuchsin;  the  entire 
cell,  excepting  the  spore,  can  be  completely, decolorized  in  acidulated  alcohol, 
and  then  methylene  blue  or  gentian  violet  applied  as  the  contrast  stain. 
We  then  have  a  blue  cell-wall  with  a  red  spore.  However,  the  beginner  is 
apt  to  be  disappointed  in  his  attempts  at  double  staining;  in  fact,  even  the 
most  skilled  bacteriological  technologists  are  apt  to  meet  with  small  success, 
and  generally  rest  satisfied  with  the  use  of  the  single  stain. 

The  pharmacist  will  have  comparatively  little  to  do  as  far  as  the  actual 
staining  of  bacteria  is  concerned.  He  should,  however,  be  able  to  prepare 
the  more  important  stains,  mordants,  and  other  solutions  which  may  be 
required  by  the  city  or  health  board  bacteriologist  or  the  physician,  and  we 
shall  therefore  give  the  more  commonly  employed  preparations. 

A.  Stock  Solutions. — Make  saturated  solutions  of  the  basic  dyes  (fuch- 
sin, gentian  violet,  and  methylene  blue)  in  95  per  cent,  alcohol.     Keep  these 
in  glass-stoppered  bottles  in  a  cool,  dark  place,  ready  for  use  in  preparing 
the  stains.     The  stock  solutions  should  in  all  intances  be  filtered  before  using. 
Secure  the  dyes  from  reliable  dealers  and  in  small  quantities.     Do  not  make 
up  large  quantities  of  stock  solutions  or  stains  proper,  as  they  gradually 
deteriorate,  particularly  if  exposed  to  light. 

B.  Mordants. — The  principal  substances  used  are  aniline,  carbolic  acid, 
tannic  acid,  glacial  acetic  acid,  ferrous  sulphate,  sodium  hydroxide  solution, 
chromic  acid,  and  a  few  others.     Those  in  general  use  are  the  two  first  named. 
The  others  have  a  more  limited  use  in  special  cases. 


72  PHARMACEUTICAL   BACTERIOLOGY. 

i.  Aniline  Water. 

Aniline,  2  c.c. 

Distilled  Water,  98  c.c. 

Shake  frequently,  and  finally  filter  several  times  through  filter  paper. 
It  should  be  perfectly  clear.  This  preparation  deteriorates  rapidly.  Make 
up  small  amounts  and  keep  in  a  dark  place.  It  becomes  .worthless,  even 
when  observing  all  precautions,  in  a  few  weeks. 

2.  Carbolic  Acid- Solution. 

Carbolic  Acid,  20  c.c. 

Distilled  Water,  100  c.c. 

Filter.     This  mordant  is  rarely  used  by  itself. 

C.  Stains. — We  give  here  the  more  important  stains,  approximately  in 
the  order  of  preferred  use. 

i.  Loeffler's  Methylene  Blue. 

Stock  Solution  (saturated)  Methylene  Blue,  30  c.c. 

i  :  10,000  Sol.  KHO  in  Dist.  Water,  100  c.c. 

Mix,  shake,  filter.  This  stain  is  much  used  as  a  general  bacterial  stain 
and  in  the  examination  of  blood,  pus,  etc. 

2.  Aniline  Gentian-Violet. 

Aniline  Water,  75  c.c. 

Stock  Solution  Gentian-Violet,  25  c.c. 

Mix,  shake,  filter.     This  is  an  excellent  bacterial  stain. 

3.  Carbol-Fuchsin. 

Stock  Solution  of  Basic  Fuchsin,  10  c.c. 

5  per  cent.  Sol.  Carbolic  Acid,  100  c.c. 

Mix,  shake,  filter.  This  is  one  of  the  most  useful  stains  with  the  so-called 
acid-proof  microbes.  It  is  also  a  spore  stain,  and  is  the  most  commonly  em- 
ployed stain  used  in  contrast  or  double  staining.  It  is  a  comparatively  slow 
stain,  but  is  permanent. 

4.  Gram's    Stain. 

Gram's  stain  is  used  for  diagnostic  purposes,  and  is  perhaps  the  best 
known  stain  in  the  entire  field  of  bacteriological  technic.  Its  value  depends 
upon  the  fact  that  certain  microbes,  when  stained  and  afterward  treated 


BACTERIOLOGICAL   TECHNIC.  73 

with  a  solution  of  iodine  and  washed  in  alcohol,  give  up  the  stain.  Such 
microbes  are  known  as  Gram-negative,  whereas  those  which  do  not  give  up 
the  stain  are  said  to  be  Gram-positive. 

The  method  of  using  this  stain  is  somewhat  complicated,  requires  care, 
and,  with  a  beginner,  often  yields  disappointing  results.  Keeping  in  mind 
the  following  will  minimize  the  disappointments: 

a.  Long-continued  (one  year  or  more)  subcultures  frequently  lose  the 
Gram-stain  behavior. 

b.  Old  cultures,  that  is,  those  which  have  been  growing  in  the  same 
medium  for  several  days  or  more,  as  a  rule  do  not  stain  characteristically. 
With  such  cultures  the  results  are  often  neither  negative  nor  positive,  just 
enough  to  be  confusing  and  perplexing. 

c.  The  solutions  used  must  be  fresh.     The  gentian-aniline  solution,  as 
well  as  the  iodine  solution,  deteriorates  quite  rapidly. 

d.  Do  not  overstain,  and  do  not  decolorize  too  long.     Stop  decolorizing 
as  soon  as  no  more  violet  color  comes  away. 

In  the  Gram  method  two  solutions  are  used,  namely: 

1.  Aniline  gentian-violet,  and 

2.  Gram's  iodine  solution. 

Iodine,  i  gm. 

Potassium  Iodide,  2  gm. 

Distilled  Water,  300  c.c. 

The  method,  briefly  outlined,  is  as  follows: 

a.  Spread  the  bacteria  evenly  and  thinly  over  the  cover-glass  (the  usual 
smear  preparation).     Stain   with    the  aniline  gentian-violet  for  from  two 
to  five  minutes.     Warming  will  hasten  and  intensify  the  staining.     Wrash 
in  water  to  remove  excess  of  stain. 

b.  Drop  on  the  iodine  solution  and  allow  it  to  act  for  about  one  minute 
or  until  the  preparation  assumes  a  coffee-brown  color.     It  may  be  desirable 
to  apply  the  iodine  a  second  time. 

c.  Wash  off  the  excess  of  iodine  in  water  and  then  decolorize  by  dropping 
on  95  per  cent,  alcohol.     Tip  the  slide  and  allow  alcohol  to  run  over  the 
preparation;  continue  until  the  violet  color  ceases  to  stream  away. 

d.  Finally  rinse  in  water  and  examine  in  water.     If  desired,  dry  and 
mount  permanently  in  Canada  balsam  or  some  other  suitable  mounting 
medium. 

e.  A  contrast  stain,  such  as  eosin,  fuchsin,  safranin,  or  Bismarck  brown, 
may  be  used,  following  (c). 

Keeping  in  mind  the  difficulties  already  referred  to  in  using  the  Gram 
method,  and  the  additional  possible  source  of  error  due  to  the  fact  that  one 
and  the  same  microbe  will  stain  but  feebly  at  one  time  and  very  intensely 


74  PHARMACEUTICAL   BACTERIOLOGY. 

at  another  time,  we  now  name  the  principal  organisms  which  are  Gram- 
positive  or  Gram-negative. 

Bacteria  and  other  Organisms  Stained  by  the  Gram  Method. 
Staphylococcus  pyogenes  aureus. 
Staphylococcus  pyogenes  albus. 
Streptococcus  pyogenes. 
Micrococcus  tetragenus. 
Micrococcus  lanceolatus. 
Bacillus  diphtheriae. 
Bacillus  tuberculosis. 
Bacillus  6f  anthrax. 
Bacillus  of  tetanus. 
Bacillus  of  leprosy. 
Bacillus  aerogenes  capsulatus. 
Oidium  albicans. 
Actinomyces  (of  actinomycosis). 

Bacteria  not  Stained  by  the  Gram  Method 
Diplococcus  of  meningitis  (intracellular) . 
Diplococcus  of  gonorrhea. 
Micrococcus  melitensis. 
Bacillus  of  chancroids  (Ducrey's). 
Bacillus  of  dysentery  (Shiga's). 
Bacillus  of  typhoid  fever. 
Bacillus  of  bubonic  plague. 
Bacillus  of  influenza. 
Bacillus  coli  communis. 
Bacillus  pyocyaneus. 
Bacillus  of  Friedlander. 
Bacillus  proteus. 
Bacillus  pyocyaneus. 
Bacillus  mallei  (glanders). 
Spirillum  of  Asiatic  cholera. 
Spirillum  of  relapsing  fever. 
Bacillus  of  pneumonia. 

5.  Pappenheim's  Stain. 

Sat.  Aqueous  Sol.  Methyl  Green,  50  c.c. 

Sat.  Aqueous  Sol.  Pyronine,  15  c.c. 

Mix  and  filter.  This  is  much  used  for  staining  bacteria  in  pus  and 
other  pathological  secretions.  The  bacteria  are  stained  a  bright  red,  while 
the  cell  nuclei  are  blue  to  purple. 

6.  Smith's  Stain. 

Stock  Sol.  Basic  Fuchsin,  10  c.c. 
Methyl  Alcohol, 

Formaldehyde,  each     10  c.c. 

Distilled  Water,  to  make  100  c.c. 


BACTERIOLOGICAL    TECHNIC.  75 

Mix  and  filter.  Let  stand  for  twenty-four  hours  before  using.  Renew 
in  three  weeks.  This  stain  is  much  used  to  distinguish  between  bacteria 
and  nuclear  substances.  Allow  the  stain  to  act  for  from  two  ta  tea. minutes. 

7.  Flagella  Staining. 

Care  is  necessary  in  staining  flagellae.  Numerous  methods  have  been 
recommended,  but  Pitfield's  method,  as  modified  by  Muir,  is  perhaps  the 
best  and  at  the  same  time  comparatively  simple.  The  following  solutions 
are  required: 

a.  Mordant. 

Tannic  Acid  (10  per  cent.  Aq.  Sol.),  .                              10  c.c. 

Sat.  Aq.  Sol.  Mercuric  Chlor.,  5  c.c. 

Sat.  Aq.  Sol.  Alum,  5  c.c. 

Carbol-Fuchsin,  5  c.c. 

Mix,  shake,  filter  or  centrifuge.  This  solution  does  not  keep  longer 
than  one  week. 

b.  Stain. 

Sat.  Aq.  Sol.  Alum,  10  c.c. 

Stock  Sol.  Gentian-Violet,  2  c.c. 

Mix,  filter.     Carbol-fuchsin  may  be  used  instead  of  gentian-violet.     This 
stain  will  not  keep  longer  than  a  few  days. 
The  method  is  as  follows: 

1.  Drop  on  mordant.     Leave  for  one  minute,  with  gentle  heat. 

2.  Rinse  in  water  for  two  minutes. 

3.  Dry  carefully  at  slight  warmth. 

4.  Stain  for  one  minute  with  gentle  heat. 

5.  Wash,  dry,  and  mount  in  Canada  balsam. 

In  making  the  cover-glass  preparation,  take  a  loopful  from  a  young 
aqueous  subculture  of  some  motile  bacillus  and  touch  it  on  the  carefully 
cleaned  cover  and  allow  the  drop  to  spread  by  rotating  and  tilting  the  cover. 
Do  not  use  the  loop  more  than  is  necessary.  Flagellae  are  very  delicate  and 
easily  destroy*  d.  Dry  very  carefully,  and  do  not  pass  through  flame  more 
than  three  times. 

8.  Spore  Staining. 

As  already  stated,  spores  (endospores)  of  microbes  stain  with  great  diffi- 
culty, for  which  reason  a  contrast  is  effected  negatively;  that  is,  the  rest  of  the 
cell  is  quickly  stained,  leaving  the  unstained,  highly  refractive  spore  to  appear 
like  a  bit  of  glass  within  the  colored  frame.  This  is  in  many  ways  the  most 
satisfactory  way  of  demonstrating  the  presence  of  spores.  The  spores  may, 


76  PHARMACEUTICAL   BACTERIOLOGY. 

however,  be  stained  by  the  usual  acid-fast  or  acid-proof  methods,  care  being 
observed  in  decolorizing.  Stain  with  hot  carbol-fuchsin  for  a  few  minutes, 
wash,  and  decolorize  quickly  with  3  per  cent,  hydrochloric  acid  in  95  per  cent, 
alcohol,  and  then  use  a  contrast  stain,  as  gentian-violet  or  methylene  blue. 
The  red  spores  will  then  appear  in  the  violet  or  blue  frame. 

9.  Capsule  Staining. 

The  gelatinous  capsule  of  microbes  is  also  stained  with  great  difficulty, 
and  requires  special  methods  and  experience  to  yield  anything  like  satisfactory 
results.  The  methods  of  Welch  and  Hiss  are  quite  satisfactory.  The  cap- 
sule is,  however,  generally  visible  without  any  staining  because  of  the  light 
contrast  that  naturally  exists.  Certain  substances,  as  glacial  acetic  acid 
(Welch  method),  cause  the  capsule  to  enlarge  and  take  up  the  stain  more 
readily.  Certain  staining  methods  bring  out  the  capsule  of  certain  mi- 
crobes, as,  for  example,  the  Gram  method  as  applied  to  pneumonia  sputum. 

The  Muir  method  is  perhaps  the  best  for  capsule  staining.  It  is  as 
follows: 

1.  Stain  in  carbol-fuchsin  for  one-half  minute,  with  gentle  heat. 

2.  Wash  lightly  in  alcohol  (95  per  cent.). 

3.  Wash  well  in  water. 

4.  Flood  with  mordant  of 

Sat.  Aq.  Sol.  Mercuric  Chlor.,  2  c.c. 

Tannic  Acid  (20  per  cent.  Aq.  Sol.),  2  c.c. 

Sat.  Aq.  Sol.  Potassium  Alum,  5  c.c. 

5.  Wash  in  water. 

6.  Wash  in  95  per  cent,  alcohol,  one  minute. 

7.  Wash  in  water. 

8.  Stain  with  methylene  blue  for  one-half  minute. 

9.  Dehydrate  in  alcohol. 

10.  Clear  in  xylene,  and  mount  in  Canada  balsam. 

There  are  numerous  other  special  stains  and  special  staining  methods, 
which  need  not  be  mentioned  here.  Should  the  pharmacist  be  called  upon 
to  prepare  any  of  these,  he  will  find  full  particulars  in  any  standard  work 
on  medical  bacteriology. 

9.  Studying  Bacteria. 

The  complete  study  of  any  one  species  of  microbe  with  a  view  to  deter- 
mining its  identity  is  a  long  and  tedious  process.  It  involves  a  study  of  the 
organism  in  its  natural  element  and  in  artificial  culture  media,  and  its 
behavior  in  animal  inoculation  tests,  etc.  Special  apparatus,  experimental 
animals  (as  rats,  mice,  guinea-pigs,  dogs,  etc.),  and  technical  experience  and 


BACTERIOLOGICAL    TECHNIC.  77 

skill  are  necessary.  Just  what  kind  of  observations  are  involved  in  such  study 
is  indicated  in  the  complete  method  as  outlined  by  the  Society  of  American 
Bacteriologists  (Jan.,  1908),  which  is  hereby  submitted  for  the  Benefit 
of  those  who  may  wish  to  acquaint  themselves  with  such  details.  The 
glossary  of  terms  should  be  carefully  considered  first  of  all.  The  decimal 
system  for  indicating  group  relationships  of  microbes  (Table  I)  is  most 
unique  and  is  very  convenient  for  active  workers.  Those  interested  will 
find  the  desired  explanations  of  the  methods  and  reagents  mentioned,  in  any 
of  the  larger  works  on  medical  bacteriology  and  in  bacteriological  technology. 
It  is  not  at  all  likely  that  the  pharmacist  will  ever  have  occasion  to  make  use 
of  the  special  methods  cited.  He  should  nevertheless  acquaint  himself  with 
them  sufficiently  to  comprehend  their  application  in  the  study  of  pathogenic 
bacteria. 

Our  bacteria  nomenclature  is  in  some  confusion,  and  unless  the  methods 
of  naming  bacteria  are  corrected,  the  confusion  is  certain  to  become  much 
greater.  The  trouble  lies  in  the  failure  to  define  group  or  generic  delimita- 
tions. The  present  generic  terms,  "bacillus"  and  "micrococcus,"  include 
too  many  species.  We  have  a  confusing  and  almost  incomprehensible 
array  of  synonyms,  of  which  those  applied  to  Rhizobium  mutabile  may  serve 
as  an  example.  The  different  names  that  have  been  given  to  this  organism 
may  be  arranged  as  follows: 

Pasteuracese,  Laurent. 

Bacteria,  Woronin,  1866. 

Bakteroiden,  Brunchorst  and  Frank,  1885. 

Microsymbiont,  Atkinson,  1893. 

Spores  or  gemmules,  Ward  and  Ericksson. 

Bacillus  radicicola,  Beyerinck,  1888. 

Cladochytrium  leguminosarum,  Vuellemin. 

Phytomyxa  leguminosarum,  Schroeter. 

Schinzia  leguminosarum,  Woronin. 

Rhizobium  leguminosarum,  Frank,  1890. 

Rhizobium  Frankii,  Schneider,  1892. 

Rhizobium  mutabile,  Schneider,  1902. 

Pseudomonas  radicicola,  Moore,  1905. 

The  above  synonomy  is  also  interesting  because  it  indicates  a  most  remark- 
able difference  of  opinion  regarding  the  nature  and  identity  of  this  root- 
nodule  organism.  Further,  as  the  result  of  the  wholly  inadequate  group 
delimitations  we  have  such  name-monstrosities  as  Granulobacillus  saccharo- 
butyricus  mobilis  nonliquifaciens ,  and  Micrococcus  acidi  paralactici  liquifaciens 
Halensi.  Reform  in  nomenclature  is  very  desirable,  and  it  must  come 
through  a  careful  definition  of  generic  groups  based  on  physiological  charac- 
ters, rather  than  upon  largely  morphological  characters,  as  is  done  now. 


78  PHARMACEUTICAL    BACTERIOLOGY. 

It  is  advised  that  the  pharmacist  refrain  from  experimenting  with  patho- 
genic organisms,  excepting  in  so  far  as  he  may  act  in  cooperation  with  the 
physician.  When  experimenting  with  pathogenic  organisms  the  greatest 
caution  is  necessary  to  guard  against  autoinoculation  and  the  spreading  of 
disease.  It  should  be  made  a  rule  to  treat  every  microbe  studied  as  though 
it  were  virulently  pathogenic,  capable  of  spreading  an  epidemic.  Never 
expose  a  colony  (plate  culture,  tube  culture,  etc.)  in  such  a  way  as  to  permit 
the  escape  of  the  organisms  into  the  air.  Pour  a  disinfecting  solution  (5 
per  cent,  carbolic  acid)  into  cultures  that  are  to  be  discontinued  and  then 
boil  container  and  all,  for  thirty  minutes,  before  washing  and  cleaning  the 
glassware.  Never  -forget  to  sterilize  the  platinum  needle  before  and  after 
making  an  inoculation  or  a  culture  transfer. 


DESCRIPTIVE  CHART— SOCIETY  OF  AMERICAN  BACTERIOLOGISTS.  1 

GLOSSARY  OF  TERMS. 

Agar  Hanging  Block,  a  small  block  of  nutrient  agar  cut  from  a  poured  plate,  and  placed 
on  a  cover-glass,  the  surface  next  the  glass  having  been  first  touched  with  a  loop  from 
a  young  fluid  culture  or  with  a  dilution  from  the  same.  It  is  examined  upside  down, 
the  same  as  a  hanging  rock. 

Ameboid,  assuming  various  shapes  like  an  ameba. 

Amorphous,  without  visible  differentiation  in  structure. 

Arborescent,  a  branched,  tree-like  growth. 

Beaded,  in  stab  or  stroke,  disjointed  or  semi-confluent  colonies  along  the  line  of  inoculation. 

Brief,  a  few  days,  a  week. 

Brittle,  growth  dry,  friable  under  the  platinum  needle.  . 

Bullate,  growth  rising  in  convex  prominences,  like  a  blistered  surface. 

Butyrous,  growth  of  a  butter-like  consistency. 

Chains,  short  chains,  composed  of  2  to  8  elements.  Long  chains,  composed  of  more  than 
8  elements. 

Ciliate,  having  fine  hair-like  extensions,  like  cilia. 

Cloudy,  said  of  fluid  cultures  which  do  not  contain  pseudozooglea. 

Coagulation,  the  separation  of  casein  from  whey  in  milk.  This  may  take  place  quickly 
or  slowly,  and  as  the  result  either  of  the  formation  of  an  acid  or  of  a  lab  ferment. 

Contoured,  an  irregular,  smoothly,  undulating  surface,  like  that  of  a  relief  map. 

Convex,  surface  the  segment  of  a  circle,  but  flattened. 

Coprophyl,  dung  bacteria. 

Coriaceous,  growth  tough,  leathery,  not  yielding  to  the  platinum  needle. 

Crater  if orm,  round,  depressed,  due  to  the  liquefaction  of  the  medium. 

Cretaceous,  growth  opaque  and  white,  chalky. 

Curled,  composed  of  parallel  chains  in  wavy  strands,  as  in  anthrax  colonies. 

Diastasic  Action,  same  as  Diastatic,  conversion  of  starch  into  water-soluble  substances  by 
diastase. 

Prepared  by  F.  D.  Chester,  F.  P.  Gorham,  Erwin  F.  Smith,  Committee  on 
Methods  of  Identification  of  Bacterial  Species.  Endorsed  by  the  Society  for  general  use 
at  the  annual  meeting,  January,  1908. 


.       BACTERIOLOGICAL   TECHNIC.  79 

Echinulate,  in  agar  stroke   a   growth   along   the  line   of   inoculation,    with    toothed   or 

pointed  margins;  in  stab  cultures  growth  beset  with  pointed  outgrowths. 
Effuse,  growth  thin,  veily,  unusually  spreading. 
Entire,  smooth,  having  a  margin  destitute  of  teeth  or  notches. 
Erose,  border  irregularly  toothed. 

Filamentous,  growth  composed  of  long,  irregularly  placed  or  interwoven  filaments. 
Filiform,  in  stroke  or  stab  cultures  a  uniform  growth  along  line  of  inoculation. 
Fimbriate,  border  fringed  with  slender  processes,  larger  than  filaments. 
Floccose,  growth  composed  of  short  curved  chains,  variously  oriented. 
Flocculent,  said  of  fluids  which  contain  pseudozooglea,    i.e.,  small   adherent  masses  of 

bacteria  of  various  shapes  and  floating  in  the  culture  fluid. 

Fluorescent,  having  one  color  by  transmitted  light  and  another  by  reflected  light. 
Gram's  Stain,  a  method  of  differential  bleaching  after  gentian- violet,  methyl- violet,  etc. 

The  +  mark  is  to  be  given  only  when  the  bacteria  are  deep  blue  or  remain  blue  after 

counterstaining  with  Bismarck  brown. 
Grumose,  clotted. 

Infundibuliform,  form  of  a  funnel  or  inverted  cone. 
Iridescent,  like  mother-of-pearl.     The  effect  of  very  thin  films. 
Lacerate,  having  the  margin  cut  into  irregular  segments  as  if  torn. 
Lobate,  border  deeply  undulate,  producing  lobes  (see  Undulate). 
Long,  many  weeks  or  months. 

Maximum  Temperature,  temperature  above  which  growth  does  not  take  place. 
Medium,  several  weeks. 

Membranous,  growth  thin,  coherent,  like  a  membrane. 

Minimum  Temperature,  temperature  below  which  growth  does  not  take  place. 
Mycelioid,  colonies  having  the  radiately  filamentous  appearance  of  mould  colonies. 
Napiform,  liquefaction  with  the  form  of  a  turnip. 
Nitrogen  Requirements,  the  necessary  nitrogenous  food.     This  is  determined  by  adding  to 

nitrogen-free  media  the  nitrogen  compound  to  be  tested. 
Opalescent,  resembling  the  color  of  an  opal. 

Optimum  Temperature,  temperature  at  which  growth  is  most  rapid. 
Pellicle,  in  fluid  bacterial  growth  either  forming  a  continuous  or  an  interrupted  sheet 

over  the  fluid. 

Peptonized,  said  of  curds  dissolved  by  trypsin. 
Persistent,  many  weeks,  or  months. 
Pseudozooglece,  clumps  of  bacteria,  not  dissolving  readily  in  water,  arising  from  imperfect 

separation,  or  more  or  less  fusion  of  the  components,  but  not  having  the  degree  of 

compactness  and  gelatinization  seen  in  zooglea. 
Pulvinate,  in  the  form  of  a  cushion,  decidedly  convex. 
Punctiform,  very  minute  colonies,  at  the  limit  of  natural  vision. 
Rapid,  developing  in  twenty-four  to  forty-eight  hours. 
Raised,  growth  thick,  with  abrupt  or  terraced  edges. 
Repand,  wrinkled. 

Rhizoid,  growth  of  an  irregular  branched  or  root-like  character,  as  in  B.  mycoides. 
Ring,  same  as  Rim,  growth  at  the  upper  margin  of  a  liquid  culture,  adhering  more  or  less 

closely  to  the  glass. 

Saccate,  liquefaction  the  shape  of  an  elongated  sac,  tubular,  cylindrical. 
Scum,  floating  islands  of  bacteria,  an  interrupted  pellicle  or  bacterial  membrane. 
Slow,  requiring  five  or  six  days  or  more  for  development. 
Short,  applied  to  time,  a  few  days,  a  week. 


80  PHARMACEUTICAL    BACTERIOLOGY. 

Sporangia,  cells  containing  endospores. 

Spreading,  growth  extending  much  beyond  the  line  of  inoculation,  i.e.,  several  millimeters 

or  more. 
Stratiform,  liquefying  to  the  walls  of  the  tube  at  the  top  and  then  proceeding  downward 

horizontally. 
Thermal  Death-point,  the  degree  of  heat  required  to  kill  young  fluid  cultures  of  an  organism 

exposed  for  ten  minutes  (in  thin-walled  test-tubes  of  a  diameter  not  exceeding  20  mm.) 

in  the  thermal  water-bath.     The  water  must  be  kept  agitated  so  that  the  temperature 

shall  be  uniform  during  the  exposure. 
Transient,  a  few  days. 

Turbid,  cloudy  with  flocculent  particles;  cloudy  flocculence. 
Umbonate,  having  a  button-like,  raised  center. 
Undulate,  border  wavy  with  shallow  sinuses. 
Verrucose,  growth  wart-like,  with  wart-like  prominences. 
Vermiform-contoured,  growth  like  a  mass  of  worms,  or  intestinal  coils. 
Villous,  growth  beset  with  hair-like  extensions. 
Viscid,  growth  follows  the  needle  when  touched  and  withdrawn,  sediment  on  shaking  rises 

as  a  coherent  swirl. 
Zooglea,  firm  gelatinous  masses  of  bacteria,  one  of  the  most  typical  examples  of  which  is 

the  Streptococcus  mesenteroides  of  sugar  vats  (Leuconostoc  mesenterioides} ,  the  bacterial 

chains  being  surrounded  by  an  enormously  thickened  firm  covering  inside  of  which 

there  may  be  one  or  many  groups  of  the  bacteria. 

NOTES. 

(r)  For  decimal  system  of  group  numbers  see  Table  i.  This  will  be  found  useful  as 
a  quick  method  of  showing  close  relationships  inside  the  genus,  but  is  not  a  sufficient 
characterization  of  any  organism. 

(2)  The  morphological  characters  shall  be  determined  and  described  from  growths 
obtained  upon  at  least  one  solid  medium  (nutrient  agar)  and  in  at  least  one  liquid  medium 
(nutrient  broth).     Growths  at  37°  C.  shall  be  in  general  not  older  than  twenty-four  to 
forty-eight  hours,  and  growths  at  20°  C.  not  older  than  forty-eight  to  seventy-two  hours . 
To  secure  uniformity  in  cultures,  in  all  cases  preliminary  cultivation  shall  be  practised  as 
described  in  the  revised  Report  of  the  Committee  on  Standard  Methods  of  the  Laboratory 
Section  of  the  American  Public  Health  Association,  1905. 

(3)  The  observation  of  cultural  and  bio-chemical  features  shall  cover  a  period  of  at 
least  fifteen  days  and  frequently  longer,  and  shall  be  made  according  to  the  revised  Stand- 
ard Methods  above  referred  to.     All  media  shall  be  made  according  to  the  same  Standard 
Methods. 

(4)  Gelatin  stab  cultures  shall  be  held  for  six  weeks  to  determine  liquefaction. 

(5)  Ammonia  and  indol  tests  shall  be  made  at  end  of  tenth  day,  nitrate  tests  at  end 
of  fifth  day. 

N 

(6)  Titrate  with        NaOH,  using  phenolphthalein  as  an  indicator:  make  titrations  at 

20 

same  time  from  blank.     The  difference  gives  the  amount  of  acid  produced. 

The  titrations  should  be  done  after  boiling  to  drive  off  any  CO2  present  in  the  culture 

(7)  Generic  nomenclature  shall  begin  with  the  year  1872  (Cohen's  first  important 
paper). 

Species  nomenclature  shall  begin  with  the  year  1880  (Koch's  discovery  of  the  poured 
plate  method  for  the  separation  of  organisms). 

(8)  Chromogenesis  shall  be  recorded  in  standard  color  terms. 


BACTERIOLOGICAL  TECHNIC.  8 1 

TABLE  i. 

A  NUMERICAL  SYSTEM  OF  RECORDING  THE  SALIEXT  CHARACTERS  OF  AN  ORGANISM 

(GROUP  NUMBER). 

100 Endospores  produced. 

200 Endospores  not  produced. 

10 Aerobic  (Strict). 

20 Facultative  anaerobic. 

30 Anaerobic  (Strict). 

i Gelatin  liquefied. 

2 Gelatin  not  liquefied. 

o.i Acid  and  gas  from  dextrose. 

o .  2 Acid  without  gas  from  dextrose. 

0.3 No  acid  from  dextrose. 

0.4 No  growth  with  dextrose. 

.01 Acid  and  gas  from  lactose. 

.02 Acid  without  gas  from  lactose. 

.03 No  acid  from  lactose. 

.04 No  growth  with  lactose. 

.001 Acid  and  gas  from  saccharose. 

.002 Acid  without  gas  from  saccharose. 

.003 No  acid  from  saccharose. 

.004 No  growths  with  saccharose. 

.0001 Nitrates  reduced  with  evolution  of  gas. 

.0002. Nitrates  not  reduced. 

.  0003 Nitrates  reduced  without  gas  formation. 

.0000  r Fluorescent. 

.00002 Violet  chromogens. 

.00003 Blue  chromogens. 

.00004 Green  chromogens. 

.  00005  ••••••••  Yellow  chromogens. 

.  00006 Orange  chromogens. 

.  00007 Red  chromogens. 

.00008 Brown  chromogens. 

.  00009 Pink  chromogens. 

.  ooooo Non-chromogenic. 

.000001 Distasic  action  on  potato  starch,  strong. 

.000002 Distasic  action  on  potato  starch,  feeble. 

.000003 Distasic  action  on  potato  starch,  absent. 

.0000001 Acid  and  gas  from  glycerin. 

.0000002 Acid  without  gas  from  glycerin. 

.  0000003 No  acid  from  glycerin. 

.  0000004 No  growth  with  glycerin. 

The  genus  according  to  the  system  of  Migula  is  given  its  proper  symbol  which  precedes 
the  number  thus:  (7) 

BACILLUS  COLI  (Esch.)  Mig.  becomes B.       222.111102 

BACILLUS  ALCALIGENES  Petr.  becomes    B.       212.333102 

PSEUDOMONAS  CAMPESTRis  (Pam.)  Sm.  be- 

comes Ps.      211 .333151 

BACTERIUM  SUICIDA  Mig.  becomes    Bact.  222.232103 

6 


82  PHARMACEUTICAL   BACTERIOLOGY. 

DETAILED  FEATURES. 

NOTE. — Underscore  required  terms.     Observe  notes  and  glossary  of  terms. 
I.      MORPHOLOGY  (2) 

i.      Vegetative  Cells,  Medium  used temp age days 

Form,  round,  short  rods,  long  rods,  short  chains,  long  chains,  filaments,  commas,  short 
spirals,  long  spirals,  clostridium,  cuneate,  clavate,  curved. 

Limits  of  Size 

Size  of  Majority 

Ends,  rounded,  truncate,  concave. 


(  Orientation  (grouping) , 


Agar  J  Chains  (No.  of  elements) 

Hanging-block  1  Short  chains,  long  chains. 

^  Orientation  of  chains,  parallel,  irregular. 

2.  Sporangia,  medium  used temp age days 

Form,  elliptical,  short  rods,  spindled,  clavate,  drum-sticks. 
Limits  of  Size Size  of  Majority 

A  f  Orientation  (grouping) 

Hanging-block      Chains  <N°-  of  elements)  '  ' 

[  Orientation  of  Chains,  parallel,  irregular. 

Location  of  Endospores,  central,  polar. 

3.  Endospores. 

Form,  round,  elliptical,  elongated. 

Limits  of  Size 

Size  of  Majority 

Wall,  thick,  thin. 

Sporangium  wall,  adherent,  not  adherent. 

Germination,  equatorial,  oblique,  polar,  bipolar,  by  stretching. 

4.  Flagella,  No Attachment,  polar,  bipolar,  peritrichiate.     How  Stained 

5.  Capsules,  present  on 

6.  ZooglecB,  Pseudozooglea. 

7.  Involution  Forms,  on in days  at °  C. 

8.  Staining  Reactions. 

1:10  watery  fuchsin,  gentian-violet,    carbol-fuchsin.     LoefBer's   alkaline   methylene 

blue. 
Special  Stains 

Gram Glycogen 

Fat Acid-fast 

Neisser 

II.    CULTURAL  FEATURES  (3) 
i.    A  gar  Stroke. 

Growth,  invisible,  scanty,  moderate,  abundant. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose,  arborescent,  rhisoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 

Luster,  glistening,  dull,  cretaceous. 

Topography,  smooth,  contoured,  rugose,  verrucose. 

Optical  Characters,  opaque,  translucent,  opalescent,  iridescent. 

Chromogenesis  (8) 

Odor,  absent,  decided,  resembling 

Consistency,  slimy,  buytrous,  viscid,  membranous,  coriaceous,  brittle. 
Medium  grayed,  browned,  reddened,  blued,  greened. 


BACTERIOLOGICAL   TECHNIC.  83 

2.  Potato. 

Growth,  scanty,  moderate,  abundant,  transient,  persistent. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose,  arborescent,  rhizoid. 
Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Luster,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

Chromogenesis  (8) Pigment  in  water  insoluble,  soluble;  other   sol- 
vents   

Odor,  absent,  decided,  resembling 

Consistency,  slimy,  butyrous,  viscid,  membranous,  coriaceous,  brittle. 
Medium  grayed,  browned,  reddened,  blued,  greened. 

3.  Loeffler's  Blood-serum. 

Stroke  invisible,  scanty,  moderate,  abundant.     Form  of  growth,  filiform,  echinulate, 

beaded,  spreading,  plumose,  arborescent,  rhizoid. 
Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Luster,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

Chromogenesis  (8) 

Medium  grayed,  browned,  reddened,  blued,  greened. 

Liquefaction  begins  in d,  complete  in d. 

4.  A  gar  Stab. 

Growth,  uniform,  best  at  top,  best  at  bottom;  surface  growth  scanty,  abundant;  restricted 

widespread. 
Line  of  puncture,  filiform,  beaded,  papillate,  villous,  plumose,  arborescent:  liquefaction. 

5.  Gelatin  Stab. 

Growth,  uniform,  best  at  top,  best  at  bottom. 

Line  of  puncture,  filiform,  beaded,  papillate,  villous,  plumose,  arborescent. 

Liquefaction,   crateriform,  napiform,   infundibuliform,   saccate,   stratiform;  begins  in 

d,  complete  in d. 

Medium  fluorescent,  browned 

6.  Nutrient  Broth. 

Surface  growth,  ring,  pellicle,  occulent,  membranous,  none. 

Clouding,  slight,  moderate,  strong;  transient,  persistent;  none;  fluid  turbid. 

Odor,  absent,  decided,  resembling 

Sediment,  compact,  occulent,  granular,  flaky,  viscid  on  agitation,  abundant,  scant. 

7.  Milk. 

Clearing  without  coagulation. 
Coagulation  prompt,  delayed,  absent. 

Extrusion  of  whey  begins  in days. 

Coagulum  slowly  peptonized,  rapidly  peptonized. 

Peptonization  begins  on d,  complete  on d. 

Reaction,  id ,  26. ,  4d ,  lod ,  2od 

Consistency,  slimy,  viscid,  unchanged. 
Medium  browned,  reddened,  blued,  greened. 
Lab  ferment,  present,  absent. 

8.  Litmus  Milk. 

Acid,  alkaline,  acid  then  alkaline,  no  change. 
Prompt  reduction,  no  reduction,  partial  slow  reduction. 

9.  Gelatin  Colonies. 
Growth  slow,  rapid. 

Form,  punctiform,  round,  irregular,  ameboid,  mycelioid,  filamentous,  rhizoid. 


»4  PHARMACEUTICAL    BACTERIOLOGY. 

Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  crateriform  (liquefying). 

Edge,  entire,  undulate,  lobate,  erose,  lacerate,  fimbriate  filamentous,  floccose,  curled. 

Liquefaction,  cup,  saucer,  spreading. 

10.  A  gar  Colonies. 

Growth  slow,  rapid  (temperature   ). 

Form,  punctiform,  round,  irregular,  ameboid,  my celioid,  filamentous,  rhizoid. 
Surface  smooth,  rough,  concentrically  ringed,  radiate,  striate. 
Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  umbonate. 
Edge,  entire,  undulate,  lobate,  erose,  lacerate,  fimbriate,  floccose,  curled. 
Internal  structure,  amorphous,  finely-,  coarsely-granular,  grumose,  filamentous,  floccose, 
curled. 

11.  Starch  Jelly. 
Growth,  scanty,  copious. 

Diastasic  action,  absent,  feeble,  profound. 
Medium  stained 

12.  Silicate  Jelly  (Fermi's  Solution). 
Growth  copious,  scanty,  absent. 
Medium  stained 

1 3 .  Cohn's  Solution. 

Growth  copious,  scanty,  absent. 
Medium  fluorescent,  non-fluorescent. 

14.  Uschinsky's  Solution. 
Growth  copious,  scanty,  absent. 
Fluid  viscid,  not  viscid. 

15.  Sodium  Chloride  in  Bouillon. 
Per  cent,  inhibiting  growth. 

1 6.  Growth  in  Bouillon  over  Chloroform,  unrestrained,  feeble,  absent. 

17.  Nitrogen.     Obtained  from  peptone,  asparagin,  glycocoll,  urea,  ammonia  salts,  nitrogen. 

18.  Best  media  for  long-continued  growth 

19.  Quick  tests  for  differential  purposes 


III.  PHYSICAL  AND  BIOCHEMICAL  FEATURES. 


1 

i.  Fermentation  tubes  con-; 
taining  peptone-water  or  Dextrose  Saccharose 
sugar-free  bouillon  and' 

Lactose 

Maltose 

Glycerin 

Mannit 

Gas  production,  in  per  cent. 

Q 

Growth  in  closed  arm 

Amount  of  acid  produced  id 


Amount  of  acid  produced  2d. 

Amount  of  acid  produced  3d. 

BACTERIOLOGICAL   TECHNIC.  85 

2.  Ammonia  production,  feeble,  moderate,  strong,  absent,  masked  by  acids. 

3.  Nitrates  in  nitrate  broth. 
Reduced,  not  reduced. 

Presence  of  nitrites ammonia 

Presence  of  nitrates free  nitrogen 

4.  Indol  production,  feeble,  moderate,  strong. 

5.  Toleration  of  Acids:  Great,  medium,  slight. 
Acids  tested. 

6.  Toleration  of  NaOH:  Great,  medium,  slight. 

7.  Optimum  reaction  for  growth  in  bouillon,  stated  in  terms  of  Fuller's  scale 

8.  Vitality  on  culture  media:  Brief,  moderate,  long. 

9.  Temperature  relations: 

Thermal  death-point  (10  minutes'  exposure  in  nutrient  broth  when  this  is  adapted 

to  growth  of  organism) C. 

Optimum  temperature  for  growth C.:  or  best  growth  at  15°  C.,  20°  C.,  25°- 

C.,  30°  C.,  37°  C.,  40°  C.,  50°  C.,  60°  C. 

Maximum  temperature  for  growth C 

Minimum  temperature  for  growth C. 

10.  Killed  readily  by  drying:  resistant  to  drying. 

11.  Per  cent,  killed  by  freezing  (salt  and  crushed  ice  or  liquid  air) 

12.  Sunlight:  Exposure  on  ice  in  thinly  sown  agar  plates,  one-half  plate  covered  (times  15 

minutes),  sensitive,  not  sensitive. 
Per  cent,  killed 

13.  Acids  produced 

14.  Alkalies  produced 

15.  Alcohols 

1 6.  Ferments:  Pepsin,  trypsin,  diastase,  invertase,  pectase,  cytase,  tyrosinase,  oxidase,  per- 

oxidase,  lipase,  catalase,  glucase,  galactase,  lab,  etc -. 


17.  Crystals  formed .... 

1 8.  Effect  of  germicides: 


Substance                  Method  used         Minutes 

Temper- 
ature 

Killing 
quantity 

Amt.  required  to 
restrain  growth 

IV.  PATHOGENICITY. 

1.  Pathogenic  to  animals. 

Insects,  crustaceans,  fishes,  reptiles,  birds,  mice,  rats,  guinea-pigs,  rabbits,  dogs,  cats, 
sheep,  goats,  cattle,  horses,  monkeys,  man. 

2.  Pathogenic  to  Plants: 


3.  Toxins,  soluble,  endotoxins. 

4.  Non-toxin  forming. 

5.  Immunity  bactericidal. 

6.  Immunity  non-bactericidal. 

7.  Loss  of  virulence  on  culture-media:  Prompt,  gradual,  not  observed  in months. 


86 


PHARMACEUTICAL   BACTERIOLOGY. 


BRIEF  CHARACTERIZATION. 

Mark  +  or  O,  and  when  two  terms  occur  on  a  line  erase  the  one  which  does  not 
apply  unless  both  apply. 


Diameter  over  T.JJL 

Chains,  filaments : 

Endospores ! 

Capsules 

Zooglea,  Pseudozooglea I 

Motile 

Involution  forms. . 

: . ! 

i  Gram's  Stain J 

Cloudy,  turbid , . .' 

Ring '...'. 

Jtsrotn  ...  . 

Pellicle i 

Sediment ; 

Shining .- . ! 

Dull 

Wrinkled ! 

Chromogenic 

Round 

Proteus-like 

Gel.  Plate  Rhizoid 

Filamentous 

Curled 

Gel.  Stab.  ^Urfr"gr°Wlh" 

Needle-growth 

Moderate,  absent. 

Abundant. . 
!     Potato  _. 

Discolored 

Starch  destroyed 

Grows  at  37°  C 

Grows  in  Cohn's  Sol 

Grows  in  Uschinsky's  Sol 

Gelatin  (4) 

Blood-serum 

Liquefaction      _ 

Casein 

Agar,  mannan 

Acid  curd 

Milk  Rennet  curd 

Casein  peptonized 

Indol(5) ." 

Hydrogen  sulphide 

Ammonia  (6) 

Nitrates  reduced  (5) 

Fluorescent 

Luminous. . . 


BACTERIOLOGICAL   TECHNIC.  87 


Animal  pathogen,  epizoon .  . 
Plant  pathogen,  epiphyte.  .  . 


I  Soil.. 
Milk. . 


Fresh  water 

Salt  water 

Sewage 

Iron  bacterium 

Sulphur  bacterium  .... 


A.  Counting  Plate  Colonies. — If  the  colonies  in  a  Petri  dish  culture  are 
few,  not  exceeding  fifty  to  one  hundred,  they  may  readily  be  counted  in  full. 
If  the  colonies  are  quite  numerous,  the  counting  may  be  made  easier  by 
marking  off  (by  means  of  a  grease  pencil  or  chalk)  the  bottom  of  the  plate 
into  two  right  angled  cross-lines   (quarter  sectors)   and  these  again  into 
equal  parts  (1/8  sectors).     Or  one  of  the  recommended  special  counting 
plates  may  be  used.     Either  the  square  or  circular  plate  will  answer  the 
purpose  (see  figures).     When  colonies  are  very  numerous  (200  and  more) 
in  a  plate  culture  and  quite  uniformly  distributed,  it  is  not  necessary  to 
count  them  all.     Count  the  colonies  in  a  number  of  squares  or  sector  areas 
(square  centimeters)  and  multiply  the  average  of  twenty  counts  by  the  num- 
ber of  squares  representing  the  entire  surface  area  of  the  culture  plate.     As 
a  rule  the  counting  should  be  complete,  however. 

From  the  plate  counts  it  is  possible,  by.  simple  mathematics,  to  deter- 
mine the  number  of  microbes  in  the  dilution  cultures  of  water,  milk,  tinc- 
tures, fluidextracts,  etc.,  as  has  already  been  explained. 

Studying  Plate  Colonies. — The  plate  colonies  should  be  studied  macro- 
scopically  and  also  with  the  aid  of  a  pocket  lens  and  under  the  low  power  of 
the  compound  microscope.  Place  the  dish  on  the  stage  of  the  microscope 
and  focus  upon  the  colonies  carefully  by  means  of  the  coarse  adjustment. 
Note  color,  outline  and  other  characteristics  of  the  colonies,  etc.,  as  already 
set  forth  under  tube  cultures  and  in  the  official  methods  of  the  Society  of 
Bacteriologists. 

B.  Making    Tube-cultures    (Subcultures). — Inoculate     test-tubes     (con- 
taining gelatin,  agar  or  other  media)  with  such  colonies  as  it  is  desired  to 
study  further.     This  is  done  as  follows:     Hold  the  test-tube  to  be  inoculated 
in  left  hand.     Take  up  the  platinum  needle  (straight  or  loop)  in  the  right 
hand  and  pass  the  entire  needle  and  glass  rod  (excepting  the  end  held) 
through  the  flame  of  a  Bunsen  burner  several  times;  heat  the  needle  to  a 
glowing  red  for  a  few  seconds  and  then  allow  it  to  cool  a  few  seconds.     Lift 
the  cover  of  the  Petri  dish  high  enough  to  pass  the  needle  under,  touch  end 
of  the  platinum  needle  (straight  or  loop)  on  colony  desired;  let  the  dish  cover 


88 


PHARMACEUTICAL   BACTERIOLOGY. 


drop  into  place  again;  remove  the  cotton  plug  from  test-tube  by  grasping  it 
between  two  fingers  (back  of  fingers  toward  the  test-tube) ;  -make  the  inocu- 
lation (deep  stab,  shallow  stab,  or  streak);  withdraw  needle;  replace  cotton 
plug;  hold  needle  in  flame  until  glowing  red.  To  prevent  the  sputtering  of 
the  material  on  the  end  of  the  needle,  hold  near  flame  until  dry  and  then  heat 


— I 


* 


'9 


\c 


L. 


FIG.  41. — Square  counting  plate.  Each  area  is  one  square  centimeter.  This  is  a 
simple  as  well  as  staisfactory  counting  plate  to  be  used  in  counting  Petri  dish  colonies  and 
tube  colonies  (tube  roll  cultures).  By  means  of  the  Arabic  numerals  and  the  letters  any 
one  of  the  100  areas  can  be  located  and  designated  for  demonstration  purposes. 

to  redness.  Singe  free  exposed  end  of  the  tube  cotton  plug  in  flame  to  kill 
and  remove  microbes  and  spores  on  the  outer  part  of  the  cotton.  The 
inoculated  tubes  are  then  numbered  and  incubated.  In  due  time  the  cul- 
tural characteristics  are  noted  and  the  observations  entered  in  a  suitable 
note-book. 

Subcultures  may  also  be  made  in  Petri  dishes,  on  potatoes,  in  tubes  con- 


BACTERIOLOGICAL    TECHNIC.  89 

taining  bouillon  broth,  blood  serum,  milk  and  other  media  with  or  without 
indicators. 

C.  Studying  Anaerobic  Microbes. — Some  microbes  have  anaerobic  tend- 
encies (facultative  aerobes)  and  some  are  absolutely  anaerobic  (obligative 
anaerobes).  The  deep  stab  culture  will  show  anaerobic  tendencies.  If 
such  tendency  exists,  development  will  be  more  active  near  the  bottom  of 
tube  (in  the  line  of  the  stab).  The  culturing  of  obligative  anaerobes  re- 
quires special  apparatus  though  the  methods  are  not  in  any  way  difficult. 
The  following  methods  are  used: 

a.  Deep  stab  culture.     This  has  already  been  sufficiently  explained.     It 
merely  indicates  possible  anaerobic  tendencies. 

b.  High-culture  methods.     Fill  the  tube  of  a  deep  stab  culture,  shallow 
stab  or  streak,  with  liquid  agar  or  gelatin  and  incubate  in  the  usual  way. 
The  medium  to  be  poured  must  not  be  warmer  than  is  absolutely  necessary 
to  render  it  liquid.     This  brings  out  possible  anaerobic  tendencies  to  a  more 
marked  degree  than  does  the  simple  deep  stab  culture. 

c.  Make  an  Esmarch   roll  tube  culture  as  follows:     Roll  a  dilution 


0%E%^^ 

FIG.  42. — Hanging-drop   culture,  sectional  profile  view.     These  slides  can  be  procured 
from  dealers  in  microscopical  supplies. 

gelatin  or  agar  tube  culture  (1:10,  1:100,  1:1000,  etc.)  so  that  all  of  the 
medium  (5  c.c.,  9  c.c.  to  10  c.c.)  is  spread  over  the  inner  surface  of  the  tube 
to  within  a  short  distance  of  the  cotton  plug.  Keep  on  rolling  slowly  until 
the  medium  has  set.  Roll  on  ice,  under  the  tap  water,  in  ice  water,  holding 
the  tube  at  the  proper  slant.  When  the  medium  has  set,  fill  in  the  entire 
tube  with  liquefied  gelatin  or  agar;  cool,  and  incubate.  Like  the  other 
methods  described,  this  will  show  possible  anaerobic  tendencies. 

d.  Various  methods  are  used  to  either  remove  the  air  (vacuum),  dis- 
place the  air,  or  remove  the  oxygen  from  the  air.  In  the  so-called  Buchner 
method,  potassium  hydroxide  and  pyrogallic  acid  are  used  to  take  up  the 
oxygen  of  the  air.  The  air  in  a  suitable  container  may  be  replaced  by 
hydrogen  by  means  of  a  Kipp  generator.  As  it  is  not  likely  that  the  phar- 
macist will  have  any  occasion  to  employ  these  methods  we  shall  pass  them 
by  with  this  mere  mention.  The  full  description  of  the  methods  will  be 
found  in  any  of  the  larger  works  on  medical  bacteriology  or  in  the  larger 
text-books  on  bacteriological  technic. 

D.  Microscopical  Examination  of  Microbes. — The  compound  microscope 
is  used  in  examining  hanging-drop  cultures,  water  mounts  and  cover-glass 
preparations.  To  make  a  hanging-drop  culture,  hollow  ground  slides 


90  PHARMACEUTICAL   BACTERIOLOGY. 

(concave  center)  are  required.  Touch  a  small  drop  of  the  culture  to  be 
examined  on  the  center  of  a  clean  and  heat-sterilized  cover-glass,  by  means 
of  a  heat-sterilized  platinum  wire  loop.  Smear  a  little  plain  petrolatum 
around  the  rim  of  the  concavity  of  the  slide  and  invert  the  cover-glass  prep- 
aration upon  the  slide,  pressing  it  gently  in  place  on  the  petrolatum. 


10  ^-r  —  *7T, 


FIG.  43.- 


-Jeffer's  circular  counting  plate  forPetri  dish  cultures.     The  entire  area  (100  sq. 
cm.)  is  marked  off  into  ten  equal  sectors  of  ten  sq.  cm.  each. 


Examine  for  a  period  of  several  hours  or  longer  as  may  be  desired.  Cell 
division,  spore  formation,  etc.,  can  be  studied  very  conveniently.  Observa- 
tions on  the  effects  of  temperature  and  rate  of  septation  may  be  made.  The 
hanging-block  preparation  is  made  by  touching  the  surface  of  a  cube  of 
nutrient  agar  with  the  bacteria  and  then  applying  this  bacterial  side  against 


BACTERIOLOGICAL    TECHNIC.  9 1 

the  cover-glass  and  mounting  like  the  hanging  drop.  The  bacteria  will  be 
found  close  to  the  cover-glass. 

Bacteria  can  be  examined  mounted  in  water  on  a  slide  coveredjvith  cover- 
glass,  in  order  to  make  observations  regarding  motility.  Of  course  it  is  not 
desirable  to  examine  pathogenic  microbes  in  this  manner  because  of  the  pos- 
sibility of  infection.  In  any  case,  great  care  should  be  observed  in  making 
the  mounts.  The  slides,  covers  and  needle  used  must  be  sterilized,  every 
antiseptic  precaution  must  be  observed;  and  avoid  placing  an  excess  of  the 
material  on  the  slide.  As  soon  as  the  observation  is  completed  (few  minutes 
to  half  an  hour)  the  mount  (slide  cover  and  all)  should  be  placed  in  a  5  per 
cent,  solution  of  carbolic  acid  preparatory  to  cleaning. 

Cover-glass  preparations,  temporary  and  permanent,  are  made  as 
follows: 

a.  Clean  a  cover-glass  thoroughly,  dry  it  well  and  heat  it.     The  heating 
will  cause  the  smear  to  spread  better  and  to  adhere  better.     The  slides  to 
be  used  must  also  be  clean  and  dry. 

b.  By  means  of  the  platinum  needle,  spread  a  bit  of  the  bacterial  growth 
or  culture  over  the  greater  portion  of  the  surface  of  the  cover-glass.     Add  a 
droplet  of  water,  if  desired,  to  separate  the  bacteria  more.     Spread  evenly. 
Do  not  use  too  much  material,  as  it  will  make  an  unsightly  mount. 

c.  Air-dry  the  smear  preparation.     This  requires  but  little  time,  perhaps 
a  minute  or  two. 

d.  Pass  the  cover-glass  preparation  through  the  flame  of  a  Bunsen  burner 
four  times.     This  must  not  be  done  too  slowly  as  that  will  char  or  burn  the 
microbes,  nor  yet  too  quickly,  as  that  would  not  coagulate  the  albuminous 
matter  and  thus  fail  to  fix  the  microbes  upon  the  cover-glass.     A  little  ex- 
perience will  soon  teach  the  proper  speed.     Four  seconds,  or  a  little  less,  is 
the  average  time  in  which  to  make  the  four  passages  through  the  flame. 

e.  Place  a  drop  or  two  of  the  stain  on  the  fixed  smear  and  allow  it  to  act 
long  enough  to  stain  sufficiently,  holding  the  cover-glass  over  a  flame  to 
warm  the  preparation.     Do  not  heat  it  more  than  60°  to  70°  C.     On  an 
average  the  stain  will  be  sufficiently  deep  in  five  minutes.    Fuchsin  requires 
longer  time  than  does  methyl-blue  or  gentian- violet. 

f.  Wash  off  the  excess  of  the  stain  under  a  small  hydrant  stream  or  by 
means  of  a  wash  bottle,  or  by  moving  it  about  in  a  dish  of  water. 

g.  After  washing,  the  preparation  may  be  examined  as  a  temporary  water 
mount.     If  it  is  satisfactory  it  may  be  made  a  permanent  mount  by  turning 
the  cover-glass  up  again  and  allowing  the  water  to  evaporate  and  then 
mounting  in  Canada  balsam  with  xylene,  oil  of  cloves  or  some  other  diluent 
for  Canada  balsam.     Oil  of  cloves  acts  on  the  stain  for  which  reason  xylene, 
benzene  or  some  other  balsam  diluent  of  the  coal-tar  series  is  preferable. 
Special  staining  methods  have  already  been  explained.     The  above  is  a 


92  PHARMACEUTICAL    BACTERIOLOGY. 

general  method  which  will  serve  most  purposes.  It  should  be  kept  in  mind 
that  the  staining  process  shrinks  the  microbes  somewhat.  The  ordinary 
staining  methods  do  not  bring  out  the  cilia.  The  fact  that  the  microbe  is 
motile  is  evidence  that  cilia  are  present,  though  it  cannot  be  known  whether 
they  are  unipolar,  bipolar  or  general,  single  or  multiple. 


CHAPTER  VI. 
BACTERIA  IN  THE  INDUSTRIES. 

A  more  careful  study  into  the  use  made  of  bacteria  in  the  arts  and  in- 
dustries will  completely  dispel  the  generally  prevalent  opinions  regarding 
the  pernicious  nature  of  bacteria.  This  erroneous  popular  conception  was 
the  outcome  of  the  earlier  activities  in  the  study  of  disease  germs.  We  know 
that  without  the  rotting  bacteria,  higher  life,  as  we  know  it,  would  be  im- 
possible. Decayed  plants  and  animals  mixed  with  sand  constitutes  the 
soil,  the  ultimate  source  of  all  higher  life. 

i.  The  Function  of  Bacteria  in  Agriculture. 

The  exact  relationship  of  soil  bacteria  to  soil  fertility  is  not  generally 
understood,  although  it  is  well  known  that  bacteria  are  abundantly  present  in 
all  soils.  In  fact,  soil  would  be  impossible  without  bacterial  action.  The 
number  of  microbes  in  one  gram  of  dry  soil  varies  considerably,  ranging 
from  about  one  million  to  six  millions  and  more.  That  these  minute  organ- 
isms must  perform  some  important  work  is  almost  self-evident.  Recent  in- 
vestigations have  demonstrated  that  the  fertility  of  the  soil  is  approximately 
proportional  to  the  number  or  quantity  of  bacteria  present. 

We  recognize  what  is  known  as  potential  fertility  and  kinetic  soil  fertility, 
or,  in  other  words,  unavailable  and  available  fertility.  By  potential  or  un- 
available fertility,  we  mean  the  existence  in  the  soil  of  plant  foods  which  are, 
so  to  speak,  locked  up  and  cannot  be  used  by  plants  in  the  form  or  chemical 
combination  in  which  they  then  exist.  By  kinetic  fertility  we  mean  that 
condition  of  the  soil  in  which  foods  are  directly  available  to  the  plants  grow- 
ing therein.  If  all  the  plant  food  substances  occurring  in  the  soil  were  directly 
or  kinetically  available,  the  productiveness  of  the  soil  would  not  lessen  appre- 
ciably for  many  years,  say  one  hundred  years  or  more.  Why  then  is  it 
necessary  to  use  fertilizers,  to  rotate  crops,  to  rest  the  soil,  etc.,  in  a  few  years 
in  order  to  prevent  soil  exhaustion?  This  is  simply  due  to  the  fact  that  in  a 
few  years  the  crop  plants  use  up  so  much  of  the  available  food  that,  unless 
more  is  supplied,  the  crop  yield  will  grow  less  and  less  until  profitable  culture 
is  impossible. 

We  are  familiar  in  a  general  way  with  soil  exhaustion  and  the  beneficial 
influence  of  soil  tilling,  of  crop  rotation,  and  the  use  and  value  of  the  various 
fertilizers.  It  is  known  that  to  let  crop  lands  lie  fallow  for  a  season  or  two 

93 


94  PHARMACEUTICAL    BACTERIOLOGY. 

renews  the  productiveness.  As  already  indicated,  soil  exhaustion  means 
that  the  crop  plants  in  a  few  years  use  up  a  high  percentage  of  the  available 
foodstuffs.  By  proper  tillage  the  moisture  retaining  power  of  the  soil  is 
increased  and  air  is  introduced,  conditions  which  are  favorable  to  the  develop- 
ment of  soil  bacteria  which  have  the  power  of  converting  a  new  supply  of 
unavailable  plant  food  into  available  plant  foods.  When  a  fertilizer,  as 
manure  or  guano,  is  added  to  the  soil,  it  is  first  attacked  by  myriads  of  rotting 
bacteria,  which  convert  some  of  the  insoluble  organic  manure  compounds 
into  soluble  compounds,  known  as  peptones  and  albumoses.  These  are  in 
turn  converted  into  ammonia  by  other  microbes,  and  the  ammonia  is  con- 
verted into  nitric  acid  by  the  so-called  nitrifying  bacteria.  The  nitric  acid 
at  once  combines  with  potash  and  lime  in  the  soil,  forming  potassium  nitrate 
and  calcium  nitrate,  in  which  form  these  substances  are  available  as  plant 
foods. 

Eminent  scientists  declare  that  certain  bacteria  of  the  intestinal  tract  are 
absolutely  essential  to  life.  Those  bacteria  constantly  associated  with  the 
roots  of  plants  presumably  play  a  very  important  part  in  the  life  history  of 
these  plants.  The  mutually  beneficial  biological  relationships  or  associa- 
tions (mutualistic  symbiosis)  between  bacteria  and  animals  and  between 
bacteria  and  plants  are  very  numerous.  In  fact  the  antagonistic  (parasitic) 
or  objectionable  associations  are  a  decided  minority.  The  recent  investi- 
gations along  this  line  have  revealed  some  very  interesting  life  conditions, 
as  will  be  more  fully  explained  in  the  discussion  of  industrial  bacteria. 

In  green  manuring,  microbes  and  higher  fungi  cause  the  starch,  sugar, 
and  cellulose  of  the  plants  used  for  this  purpose  to  undergo  fermentation; 
organic  acids  are  liberated  which  render  the  insoluble  soil  phosphates  (of 
calcium)  soluble.  That  is,  the  insoluble  basic  phosphates  are  converted 
into  neutral  phosphates,  which  are  soluble.  Carbon  dioxide,  another  very 
important  bacterial  product,  combines  with  potash  to  form  carbonates,  and 
these  in  turn  acf  upon  the  silica  in  the  soil,  forming  the  potash  zeolites 
(hydrates  of  silica).  Certain  microbes,  lower  hyphal  fungi  and  soil  algae, 
have  the  power  of  chemically  binding  the  free  nitrogen  of  the  air,  thus  ren- 
dering this  abundant  element  available  as  plant  food. 

By  means  of  thorough  soil  cultivation  and  the  systematic  use  of  fertilizers 
we  simply  encourage  the  development  of  the  particular  microbes  that  will 
set  free  or  render  available  the  food  substances  required  by  the  crop  plants 
under  cultivation.  Agricultural  bacteriology  is  beginning  to  make  practical 
use  of  certain  plant  food  forming  microbes.  Of  these  the  free  nitrogen- 
binding  microbes  are  most  promising  from  the  standpoint  of  practical  com- 
mercial utility,  and  have  received  much  attention  in  recent  years.  The 
more  important  species  are:  Rhizobium  mutabile,  Bacillus  ellenbachiensis 
Caron,  Azotobacter  chroococcum;  Bacillus  subtilis,  Bacillus  calif orniensis,  and 


BACTERIA   IN   THE   INDUSTRIES. 


95 


FIG.  44. — Longitudinal  section  through  red  clover  rootlet,  showing  tubercle  formation 
due  to  the  root  nodule  microbe,  Rhizobium  mutabile.  The  tubercle  is  only  partially  de- 
veloped, a,  root  hairs.  These  do  not  develop  on  the  nodule,  b,  the  normal  root  par- 
enchyma, c,  vascular  tissue,  d,  infected  area,  also  showing  the  infecting  strands  (In- 
fectionsfaden).  The  cells  are  filled  with  bacteria,  e,  apical  areas,  the  growing  areas  of 
the  tubercle. 


96 


PHARMACEUTICAL    BACTERIOLOGY. 


a  few  others.     Of  these,  Rhizobium  mutabile,  the  root-nodule  bacterium  of 
the  Leguminosae,  has  received  most  attention. 

The  first  to  suggest  a  plan  for  practically  utilizing  the  root  nodule  bacteria 
(Rhizobia)  and  to  secure  letters  patent  for  the  process  in  Germany  and  the 
United  States,  were  Nobbe  and  Hiltner,  of  Tharand,  Germany.  Patent 
No.  570,876  was  granted  Nobbe  and  Hiltner  in  the  United  States,  November 
3,  1896.  This  patented  fertilizer  for  leguminous  plants  consisted  of  pure 
cultures  of  the  several  varieties  (or  perhaps  species)  of  R.  mutabile,  each  spe- 
cies of  plant,  as  bean,  pea,  clover,  alfalfa,  etc.,  having  the  cultures  derived 
from  the  root  nodules  peculiar  to  it. 


FIG.  45. — Root  nodules  of  sweet  clover,  somewhat  magnified.  A,  rootlets  with 
nodules,  a,  single  nodules,  b,  clusters  of  nodules.  These  are  sometimes  very  large, 
consisting  of  hundreds  of  nodules,  loosely  united.  B,  diagram  of  single  nodule,  a,  un- 
infected  area,  b,  infected  area. 

This  commercial  preparation  was  given  the  name  "nitragin,"  and  its 
efficiency  was  quite  carefully  and  extensively  tested  and  commented  upon 
by  European  and  American  investigators.  The  consensus  of  opinion  seems 
to  be  that  it  was  of  doubtful  practical  utility  for  agricultural  purposes. 
Some  authorities  maintained  that  it  was  of  unquestionable  value  in  virgin 
soil.  In  rich  and  otherwise  favorable  soil  conditions  it  is  of  only  slight  value. 
It  is  maintained  that  nitragin  aids  very  materially  in  developing  and  ripening 
the  fruit.  As  becomes  evident  from  careful  consideration,  the  value  of 
this  microbic  fertilizer  depends  upon  whether  or  not  it  will  cause  an  increased 
development  in  the  number  and  size  of  root  tubercles  over  and  above  those 
which  would  develop  without  the  presence  of  this  artificial  aid.  If  the  soil 


BACTERIA  IN  THE  INDUSTRIES. 


97 


is  already  well  supplied  with  rhizobia  or  root  tubercle  bacteria,  as  soil  would 
naturally  be  if  the  leguminous  plants  under  consideration  had  been  grown 
in  it  for  one  or  more  seasons,  nitragin  would  in  all  probability  be-oUittle  or 
no  value.  In  any  case,  the  anticipated  results  have  not  been  fully  realized, 
and  nitragin  is  withdrawn  from  the  market,  and  is  no  longer  manufactured. 

A  second  and  later  improvement  in  the  method  of  inoculating  seeds  with 
root  tubercle  bacteria  (Rhizobia)  is  given  by  Hartleb  in  the  specifications 
forming  part  of  letters  patent  No.  674,765,  granted  May  21,  1901,  at  Wash- 
ington, D.  C.  Although  not  so  stated  in  the  specifications,  it  is  evident  that 


0 


i 

o       0 

I 


FIG.  46. — Motile  forms  of 
Rhizobium  mutabile  as  they  ap- 
pear in  fresh  cultures.  They  are 
very  small,  1/2  to  2/3  p  in  length. 


FIG.  47. — Non-motile  matured  forms  of  R. 
mutabile  as  they  appear  in  mature  sweet  clover 
root  nodules.  Most  of  them  show  the  forked 
ends.  This  may  be  considered  the  normal  form 
of  this  organism. 


the  Hartleb  process  is  a  method  for  applying  pure  rhizobia  cultures  to  seed 
of  leguminous  plants.  Whether  the  method  offers  any  advantages  over 
the  method  of  Nobbe  and  Hiltner  is  questionable.  In  any  case  it  would 
prove  practically  advantageous  only  under  the  conditions  referred  to  under 
the  discussion  of  nitragin.  Although  the  method  has  been  freely  discussed 
and  experimented  upon  in  Germany,  the  fertilizer  is  no  longer  on  the  market. 
There  is  on  the  market  a  third  patented  germ  or  microbe  soil  fertilizer  of 
German  origin,  known  as  "alinit."  It  consists  essentially  of  a  pure  culture 
of  the  soil  bacillus  known  as  Bacillus  ellenbachiensis  alpha  or  Bacillus  ellen- 
bachiensis  Caron.  The  germ  was  first  brought  to  the  attention  of  the  agri- 
culturists by  Caron,  a  land  owner  of  Germany,  who  first  isolated  it  and 
called  attention  to  the  fact  that  it  had  the  power  of  chemically  binding  the 
free  nitrogen  of  the  air.  The  microbe  is  said  to  be  closely  allied  to  B.  mega- 
7 


98 


PHARMACEUTICAL  BACTERIOLOGY. 


FIG.  48. — R.  mutabile  as  it  appears  in  mature  nodules  of  red  and  white  clover  root 
nodules.     This  may  be  considered  the  extreme  form  variation  due  to  hyper-nutrition. 


FIG.  49. — R.  mutabile  from  the  root  nodules  of  Trifolium  heterodon,  showing  the  ex- 
treme form  variation  due  to  hyper-growth.  The  forms  shown  in  Figs.  7,  8  and  9  are 
simply  natural  involution  forms  of  the  same  species  due  to  differences  in  environment  and 
host  relationship.  The  chromatin  bodies  found  in  the  hyper-nourished  forms  (Fig.  48) 
are  probably  reserve  products. 


BACTERIA   IN    THE   INDUSTRIES.  99 

therium  and  B.  subtilis.  According  to  some  authorities  it  is  especially  con- 
cerned in  assimilating  free  nitrogen  for  gramineous  plants..  If  it  is  true  it 
may  prove  of  great  value  to  grain  growers. 

The  commercial  alinit  is  a  dry  pulverulent  substance  of  a  yellowish- 
gray  color,  with  about  10  per  cent,  moisture  and  2.5  per  cent,  nitrogen.  It 
is  evidently  prepared  by  mixing  spore-bearing  pure  cultures  of  the  bacillus 
of  Caron,  with  a  base  of  starch  and  albumen.  It  is  used  to  inoculate  soil 
either  by  spreading  it  broadcast  or  by  sowing  or  otherwise  planting  it  with 


FIG.  50.  FIG.  51. 

FIG.  50. — Involution  forms  of  R.  mutabile  as  they  occur  in  artificial  culture  (beef 
broth).  R.  mutabile  can  be  cultured  quite  readily  upon  a  great  variety  of  culture  media, 
showing  marked  adaptibility  to  variations  in  food  supply  and  in  environment. 

FIG.  51. — Azotobacter  agilis  deeply  stained.  This  organism  is  actively  motile  as 
indicated  by  the  pressure  of  numerous  cilia.  The  closely  related  species  A.  chroococcum 
is  less  actively  motile.  Both  possess  the  power  of  free  nitrogen  assimilation  to  a  high 
degree,  especially  when  cultured  in  a  nitrogen-free  medium.  The  organisms  are  large 
(3  to  6  fji  in  diameter)  in  the  comparative  sense.  Clostridium  pastorianum  is  also  an  active 
free  nitrogen  assimilator,  but  differs  from  the  Azotobacters  in  that  it  forms  spores,  a  prop- 
erty which  may  render  it  highly  valuable  in  economic  agriculture  as  cultures  in  the  sporu- 
lating  stage  can  be  kept  for  a  long  time  while  the  cultures  of  non-sporulating  bacteria  soon 
die  off  or  lose  their  potency. 

the  seed.  It  is  not  a  nodule  or  root  tubercle-forming  organism,  and  does 
not  enter  into  intimate  symbiotic  or  biologic  relationship  with  plants.  Its 
work  is  simply  that  of  binding  free  nitrogen,  forming  nitrogenous  compounds 
which  enrich  the  soil,  thus  increasing  the  yield  of  any  crop  benefited  by  such 
compounds. 

It  is  known  that  there  are  soil  bacteria  which  are  more  especially  active 
with  certain  plants  or  groups 'of  related  plants,  and  this  peculiarity  has 
suggested  the  possibility  of  isolating  them,  artificially  increasing  their  potency 
and  using  them  commercially  for  fertilizing  purposes.  It  is  also  true  that 
not  all  soil  bacteria  are  beneficent.  Under  certain  conditions,  pathogenic 
and  otherwise,  harmful  microbes  are  present  in  great  numbers  and  become 


100  PHARMACEUTICAL   BACTERIOLOGY. 

very  destructive  to  crop  plants,  causing  diseases  of  roots  and  other  plant 
organs.  Bacillus  calif orniensis,  isolated  from  sugar  beets  and  from  sugar 
beet  soil,  appears  to  promote  the  growth  of  sugar  beets,  particularly  the 
seedlings.  The  microbic  leguminous  fertilizer  of  the  Department  of  Agri- 
culture, Washington,  D.  C.,  is  a  slight  modification  of  the  Hiltner  method. 
The  microbic  cultures  are  grown  in  the  absence  of  nitrogen  or  nitrogenous 
compound  making  them  nitrogen  hungry,  thus  increasing  their  potency  to 
produce  nodules  when  brought  in  association  with  germinating  leguminous 
plants.  The  process  is  patented  in  the  United  States,  and  free  samples  have 
been  liberally  distributed  among  farmers  for  test  purposes,  but  the  results 
reported  have  been  rather  variable,  and  as  a  whole  quite  unsatisfactory. 
The  indications  are,  however,  that  future  experiments  will  clear  up  the  pres- 
ent difficulties,  and  some  of  these  so-called  vest-pocket  microbic  fertilizers 
will  no  doubt  prove  highly  beneficial. 

2.  Bacteria  in  Milk  and  in  the  Dairying  Industry. 

Bacteria  play  an  important  part  in  modern  dairying,  and  they  are  destined 
to  play  even  a  more  significant  part  in  the  near  future.     Certain  microbes 


FIG.  52. — Lactic  acid  bacillus.  There  is  a  large  group  of  bacteria,  similar  in  appear- 
ance to  the  lactic  acid  bacillus,  which  have  the  power  of  forming  lactic  acid  in  milk.  Some 
of  these  are  used  in  pure  culture  to  make  the  so-called  artificial  buttermilk.  Milk  bacte- 
riology is  still  in  its  infancy.  For  so  long  have  we  been  accustomed  to  the  use  of  contami- 
nated (filthy)  milk  that  in  a  recent  test  made  with  samples  of  pure  milk  and  samples  of  which 
cow  manure  was  added,  90  per  cent,  of  those  who  were  asked  to  taste  the  milks  preferred 
the  milk  to  which  the  cow  manure  was  added,  declaring  that  it  was  the  only  sample  which 
had  a  "milk  flavor." 

are  active  in  the  ripening  of  cream,  butter  and  cheese.  Formerly  it  was 
customary  to  let  nature  attend  to  the  inoculation  of  the  cheese,  resulting 
in  a  rather  variable  product.  Now  the  up-to-date  dairy-man  inoculates  the 


BACTERIA   IN   THE   INDUSTRIES.  IOI 

cheese  with  pure  cultures  of  the  kind  of  microbe  producing  the  desired  flavor 
as  Roquefort,  Bre,  Limburger,  etc.  In  time  it  will  no  doubt  be  possible  to 
produce  hitherto  unheard-of  cheese  flavors  by  means  of  new  spectes,-varieties, 
and  strains  of  cheese  microbes.  Cream-  and  butter-flavor  bacteria  are  also 
used.  The  souring  of  milk  is  due  to  the  omnipresent  but  illy  denned  Bacillus 
acidi  lactici  and  other  bacteria.  Stringy  or  ropy  milk  is  due  to  bacterial  in- 
fection. Under  conditions  favorable  to  the  development  of  the  organisms, 
the  ropiness  appears  within  from  twelve  to  twenty-four  hours  after  milking, 
and  becomes  so  pronounced  that  the  milk  can  be  drawn  out  in  long  threads 
or  strings.  It  is  a  not  uncommon  condition  of  milk  in  Switzerland,  where  it 
is  considered  specially  noxious,  but  in  Holland  it  has  been  produced  by 
design  for  making  Edam  cheese.  Ropiness  of  milk  is  caused  by  a  variety  of 
micro-organisms,  among  them  being  Bacillus  actinobacter,  B.  lactis  viscosus, 
B.  gummosus,  etc.  The  micro-organism  used  in  Holland  for  the  manufacture 
of  the  cheese  referred  to  is  known  as  the  Streptococcus  hollandicus.  The 
Bacillus  cyanogenus  causes  the  milk  to  become  blue  without  coagulating  it  or 
rendering  it  acid.  The  Bacillus  butyricus  occurs  in  milk  which  it  coagulates, 
also  producing  butyric  acid.  It  is  this  microbe  which  develops  the  rancidity 
of  butter.  There  are,  however,  many  different  species  of  microbes  which 
produce  butyric  acid  fermentation. 

Freshly  drawn  milk  is  not  germ-free,  even  under  the  most  aseptic  and  sani- 
tary conditions  and  surroundings.  As  a  rule  even  the  milk  in  the  udder  con- 
tains some  germs,  in  spite  of  the  fact  that  milk  possesses  decidedly  bacteri- 
cidal properties.  However,  the  milk  from  different  animals  varies  in  this  re- 
gard. The  bacterial  impurities  of  freshly  drawn  milk  are  traceable  to  the 
skin  of  the  cow,  the  dust  and  filth  about  cow  stables,  the  vessel  containing 
the  milk,  and  above  all  to  the  hands  of  the  milkers.  The  milker  is  often 
the  cause  of  inoculating  the  milk  with  disease  germs,  as  typhoid,  colon 
bacillus,  diphtheria,  scarlet  fever,  small-pox,  and  tuberculosis.  The  medical 
journals  cite  cases  of  typhoid  epidemics  traceable  to  milkers  who  were  "ty- 
phoid carriers"  without  actually  suffering  from  the  disease.  Cows  are  very 
susceptible  to  tuberculosis,  and  the  milk  from  tuberculous  animals  has  in- 
fected thousands  upon  thousands  of  children  and  many  adults. 

Since  milk  is  an  excellent  culture  medium  for  a  great  variety  of  germs,  it 
is  evident  that,  under  favorable  conditions,  it  may  be  a  fruitful  source  of 
infections.  Serious  epidemics  of  typhoid  fever  and  of  diphtheria  have 
been  traceable  to  and  exactly  limited  to  the  milk  route  of  a  certain  dairy-man. 
Tuberculous  infections  of  the  children  in  a  number  of  families  have  been 
traceable  to  the  milk  from  a  single  animal.  As  a  rule  mixed  milk  (that  is  the 
milk  from  many  animals)  is  safer  than  the  milk  from  a  single  animal,  though 
this  is  not  necessarily  always  the  case.  The  milk  from  animals  that  are  free 
from  disease  and  that  are  tested  regularly  (every  six  months)  for  tuberculosis, 


102  PHARMACEUTICAL   BACTERIOLOGY. 

and  that  are  kept  under  sanitary  conditions,  is  absolutely  safe,  provided  the 
containers  are  clean  and  the  milkers  and  others  in  the  dairying  establishment 
are  free  from  latent  or  active  communicable  disease  and  are  cleanly  in  their 
habits.  The  number  of  germs  in  freshly  drawn  milk  varies  from  1000  to 
several  millions  per  c.  c.,  and  is  directly  proportional  (within  the  limits  indi- 
cated) to  the  cleanliness  and  sanitary  conditions  of  the  dairying  establish- 
ment. The  bacterial  content  of  milk  from  the  same  source  is  of  course  higher 
in  warm  and  hot  weather  than  it  is  in  cold  weather,  other  things  being  equal. 
Certain  dairying  establishments  supply  what  is  known  as  " certified  milk," 
or  milk  which  is  certified  by  the  board  of  health  as  coming  from  animals  that 
are  regularly  tested  for  tuberculosis  and  which  are  kept  under  the  sanitary 
conditions  imposed  by  the  milk  commission  or  by  the  board  of  health,  further- 
more, such  milk  must  be  bottled  in  sterilized  bottles  which  are  hermetically 
sealed  and  placed  on  ice  at  once  and  kept  on  ice  until  delivered  to  the  con- 
sumer. There  is,  however,  a  lack  of  uniformity  in  the  regulations  govern- 
ing the  supply  of  certified  milk  in  different  communities.  The  following 
conditions  should  prevail: 

a.  All  cows  should  be  healthy,  that  is,  free  from  diseases  of  all  kinds. 
The  animals  should  be  tested  for  tuberculosis  every  six  months.     As  soon 
as  an  animal  gives  a  positive  reaction  for  tuberculosis,  it  should  be  removed 
from  the  herd  and  killed.     Milk  from  sick  animals  (any  disease  whatever) 
should  not  be  used. 

b.  The  sanitary  conditions  and  environment  of  pasture,  grazing  lands, 
sheds,  stables,  etc.,  should  be  excellent.     The  entire  water  supply  should  be 
pure,  and  all  water  supplies  should  be  tested  chemically  and  bacteriologically 
at  suitable  intervals.     All  food  supply  for  cows  must  be  wholesome  and  free 
from  objectionable  contaminations. 

c.  Those  employed  about  the  establishment  must  be  free  from  latent  or 
active  disease.     They  should  be  tested  for  tuberculosis,  latent  typhoid,  and 
should  be  examined  for  skin  diseases.     They  must  be  cleanly  in  their  habits. 
Before  milking,  the  hands  of  the  milkers  and  the  teats  of  the  animals  should 
be  washed  with  clean  warm  water  and  then  dried  with  a  clean  towel. 

d.  The  containers  must  be  sterilized  thoroughly  every  day,  inside  and 
outside.     This  can  be  done  by  thoroughly  washing  and  rinsing  in  boiling 
hot  water  and  thoroughly  drying,  before  pouring  milk  into  them. 

e.  Just  as  soon  as  the  milk  is  drawn,  it  should  be  bottled  (sterilized 
bottles) ,  bottles  capped,  hermetically  sealed  (paraffin) ,  and  placed  on  ice  until 
ice-cold,  and  delivered  at  once  to  the  consumer.     The  bottles  should  be  on 
ice  in  delivery,  and,  even  though  hermetically  sealed,  should  be  kept  away 
from  dust  and  dirt.     The  bottles  should  be  placed  in  paper  bags  so  that  the 
driver  need  not  touch  them  at  all.     The  housewife  should  take  the  bottle 


BACTERIA   IN   THE   INDUSTRIES.  103 

from  the  bag  and  place  it  in  the  ice-chest,  cellar,  or  cooler  until  the  milk  is 
wanted  for  use. 

Such  certified  milk  would,  in  all  probability,  contain  less  than  1000  mi- 
crobes per  c.c.,  perhaps  not  more  than  200  to  500  per  c.c.,  whereas  most  of 
the  so-called  certified  milk  found  on  the  market  contains  from  1000  to  10,000 
and  more  microbes  per  c.c.  The  bacterial  content  of  fresh  uncertified  milk 
ranges  from  20,000  to  several  millions,  although  from  20,000  to  50,000  per 
c.c.  is  the  maximum  number  allowed  by  most  boards  of  health.  As  already 
stated,  milk  is  an  excellent  culture  medium  for  bacteria,  and  under  favorable 
temperature  conditions  the  rate  of  development  is  very  rapid.  In  the  United 
States  the  requirements  of  the  bacteriological  standardization  of  milk  are 
very  variable  and  are  rather  arbitrarily  fixed  by  the  different  boards  of 
health  that  may  insist  upon  any  standard  at  all.  In  some  cities  a  summer 
and  a  winter  standard  is  recognized.  75,000  bacteria  per  c.c.  .may  be  the 
winter  standard,  while  100,000  per  c.c.  is  the  summer  standard.  Nearly 
all  boards  of  health  admit  that  3,000,000  bacteria  per  c.c.  is  the  maximum 
number  which  may  be  permissible. 

Milk,  on  standing,  should  show  no  dirt  deposit.  This  crude  test  is  a 
fairly  reliable  guide  as  to  the  sanitary  conditions  in  the  dairying  establishment 
and  the  rules  of  cleanliness  that  are  observed.  It  has  been  shown  that  the 
quantity  of  bacteria  in  freshly  drawn  milk  is  directly  proportional  to  the 
amount  of  dirt  (sediment)  present.  A  bottle  or  tumbler  full  of  milk  should 
show  no  dirt  sediment  after  standing  for  an  hour  or  more. 

Good  cows'  milk  should  have  from  3.5  to  3.75  per  cent,  of  butter  fat.  It 
is  marketed  in  three  forms:  Full  milk  having  all  of  the  butter  fat,  half  milk 
or  partially  skimmed  milk,  and  skimmed  milk.  Because  of  the  variability 
of  milk  which  is  partially  skimmed,  it  would  be  wise  to  withdraw  it  from  the 
market.  When  milk  is  sold  without  further  specification,  full  or  unskimmed 
milk  is  understood.  It  is  unlawful  to  sell  skimmed  milk  as  milk,  or  without 
designating  it  as  skimmed  milk. 

In  some  countries,  as  Germany  for  example,  the  rules  and  regulations  di- 
rected against  dairies,  dairying  and  the  sale  of  milk,  are  very  far-reaching, 
and  are  strictly  enforced  by  the  local  health  authorities.  Specific  rules  are 
laid  down  as  to  what  milk  may  or  may  not  be  marketed,  how  the  cows  are  to 
be  kept,  what  cattle  diseases  render  the  milk  unfit  for  use,  how  cows  and  milk- 
ers must  be  prepared  for  the  milking  process,  etc.  The  use  of  preservatives 
is  not  permitted,  because  these  substances  reduce  the  digestibility  of  the 
milk  and  because  their  use  encourages  lax  and  careless  methods  in  the  dairy- 
ing establishments. 

The  bovine  disease  most  to  be  dreaded  is  tuberculosis.  It  is  very  prev- 
alent among  cattle,  and  the  milk  from  tuberculous  cows  is  a  serious  menace 
to  the  health  of  those  who  use  it,  particularly  to  susceptible  (by  inheritance) 


104  PHARMACEUTICAL   BACTERIOLOGY. 

children.  The  most  efficient  means  of  safeguarding  the  public  health  against 
this  source  of  infection  consists  in  removing  the  infected  animals  from  the 
herd,  with  a  view  of  disposing  of  them  by  slaughter  and  burial  as  soon  as  cir- 
cumstances will  permit.  Where  this  wasteful  method  has  been  employed 
the  results  have  been  discouraging,  even  when  the  State  recompensed  the 
owner  in  part  for  the  loss  of  his  stock.  The  government  meat  inspection 
regulations  admit  the  use  of  meat  of  slightly  tuberculous  animals  for  food, 
for  it  is  declared  that  under  such  circumstances  the  cooking  of  meat  is  an 
effective  safeguard  against  danger. 

Testing  cows  for  the  presence  of  latent  or  undeveloped  forms  of  tubercu- 
losis is  simple,  safe,  and  should  be  rigidly  persisted  in.  Tuberculin  is  in- 
jected into  the  neck  or  shoulder  region.  If  tuberculosis  exists  there  will  be  a 
rise  in  temperature  (102°  to  104°  F.),  in  the  course  of  from  eight  to  eighteen 
hours.  If  the  disease  is  far  advanced  there  may  be  no  reaction,  in  fact,  the 
reaction  is  then  unnecessary  as  the  indications  are  already  sufficiently  positive. 

The  tuberculin  used  is  prepared  from  glycerinated  bouillon  in  which 
tubercle  bacilli  have  been  grown  from  six  to  eight  weeks.  The  bouillon 
culture  is  first  boiled  for  two  hours  to  kill  all  the  living  organisms.  It  is  then 
filtered  under  pressure  through  a  germ-proof  earthenware  filter  to  remove 
the  dead  bodies  of  the  germs,  concentrated  by  evaporation,  a  little  carbolic 
acid  added,  and  it  is  then  bottled  for  distribution.  There  is  no  evidence  that 
its  use  causes  an  increase  in  the  rapidity  of  the  progress  of  the  disease  in 
animals  already  affected  with  tuberculosis,  or  that  it  is  injurious  to  them  in 
any  other  way.  It  does  not  even  temporarily  injure  the  quality  of  the  milk. 

Preservatives,  as  boric  acid,  salicylic  acid,  benzoic  acid,  sodium  benzoate 
and  formalin,  are  sometimes  added  to  milk  to  prevent  bacterial  development. 
A  very  small  amount  of  formalin  (i:  10,000)  is  sufficient  to  check  the  souring 
of  milk.  The  others  are  added  in  larger  amounts  (i :  1000  or  more) .  These 
additions  are  not,  as  a  rule,  appreciable  through  the  sense  of  taste  or  smell 
and  do  not  in  any  way  modify  the  appearance  of  the  milk.  In  some  countries 
milk  preservatives  are  permissible,  in  others  they  are  not,  and  in  still  others 
they  are  permitted  provided  there  is  a  declaration  to  that  effect  and  the 
amount  does  not  exceed  a  definite  percentage,  as  provided  by  law. 

In  England,  a  limited  amount  of  certain  preservatives  added  to  milk  is 
permissible,  the  argument  being  that  it  is  better  to  supply  preserved  milk  than 
milk  loaded  with  germs.  This  argument  has  its  commendable  features.  In 
very  large,  congested  cities  like  London,  New  York  and  Chicago,  it  is  im- 
possible to  supply  the  poor  with  certified  milk  or  milk  which  can  be  kept  free 
from  excessive  germ  development  until  it  is  wanted  for  consumption. 

Boiling  the  milk  for  twenty  minutes  kills  the  germs,  but  unfortunately  the 
boiling  temperature  produces  certain  changes  which  greatly  reduce  the  food 
value  of  the  milk,  besides  the  germicidal  properties  of  the  milk  are  destroyed, 


BACTERIA   IN   THE    INDUSTRIES.  105 

so  that  the  bacterial  development  is  afterward  even  more  active  than  before. 
Sterilizing  at  lower  temperature  (50°  to  80°  C.),  known  as  pasteurizing,  does 
not  interfere  with  the  nutritive  qualities  of  the  milk,  but  destroys_ihe_bacteri- 
cidal  properties,  as  already  mentioned.  The  process  is,  however,  generally 
recommended  by  physicians.  A  simple  home  method  may  be  carried  out  as 
follows  (Roger): 

Milk  is  most  conveniently  pasteurized  in  the  bottles  in  which  it  is  deliv- 
ered. To  do  this  use  a  small  pail  with  a  perforated  false  bottom.  An 
inverted  pie  tin  with  a  few  holes  punched  in  it  will  answer  this  purpose 
Punch  a  hole  through  the  cap  of  one  of  the  bottles  and  insert  a  thermometer. 
Set  the  bottles  of  milk  on  the  pie  tin  in  the  pail  and  fill  the  pail  with  water 
nearly  to  the  level  of  the  milk.  Put  the  pail  on  the  stove  or  over  a  gas  flame 
and  heat  it  until  the  thermometer  in  the  milk  shows  not  less  than  65°  C.  nor 
more  than  70°  C.  The  bottles  should  then  be  removed  from  the  water  and 
allowed  to  stand  from  twenty  to  thirty  minutes.  The  temperature  will 
fall  slowly,  but  may  be  held  more  uniformly  by  covering  the  bottles  with  a 
towel.  The  punctured  cap  should  be  replaced  with  a  new  one,  or  the 
opening  sealed  with  wax  or  paraffin,  or  the  bottle  may  be  covered  with  an 
inverted  cup. 

After  the  milk  has  been  held  as  directed  it  should  be  cooled  as  quickly 
and  as  much  as  possible  by  setting  in  water.  To  avoid  danger  of  breaking 
the  bottle  by  a  too  sudden  change  of  temperature,  this  water  should  be  warm 
at  first.  Replace  the  warm  water  slowly  with  cold  water.  After  cooling, 
milk  should  in  all  cases  be  kept  at  the  lowest  available  temperature. 

It  should  be  remembered  that  pasteurization  does  not  destroy  all  bacteria 
in  milk,  and  after  pasteurization  it  should  be  kept  cold  and  used  as  soon  as 
possible. 

Rosenau  sums  up  the  pros  and  cons  of  milk  pasteurization  as  follows: 

Advantages. — The  advantage  of  pasteurization  is  that  it  is  a  cheap  and 
effective  means  of  preventing  the  transmission  of  infectious  diseases  such  as 
tuberculosis,  typhoid  fever,  diphtheria,  scarlet  fever,  etc.,  commonly  spread 
by  milk. 

Disadvantages. — a.  Pasteurization  promotes  carelessness  on  the  farm 
and  dairy,  etc.  (This  may  be  controlled  by  proper  regulations,  inspections 
and  laboratory  examinations.) 

b.  Pasteurization  renders  milk  less  digestible.     (While  it  is  generally 
conceded  that  boiled  milk  commonly  induces  constipation,  the  majority  of 
the  evidence  plainly  indicates  that  pasteurization  has  little,  if  any,  effect  on 
the  digestibility  of  the  milk.) 

c.  Pasteurized  milk  favors  the  production  of  rickets  and  scurvy.     (There 
is  no  proof  to  this  effect  and  authorities  agree  that  the  danger  is  slight;  and, 
further,  that  it  may  readily  be  obviated.) 


106  PHARMACEUTICAL   BACTERIOLOGY. 

d.  By  destroying  the  non-spore-bearing  bacteria,  pasteurization  some- 
times allows  toxic  organisms  to  grow  and  produce  serious  poisons  in  the 
milk.     (On  the  other  hand,  these  same  poisons  are  more  frequently  pro- 
duced in  milk  that  has  not  been  pasteurized,  and  thus  danger  may  be  ob- 
viated in  pasteurized  milk  by  cooling  it  quickly,  keeping  it  cold  and  short- 
ening the  time  for  distribution.) 

e.  Pasteurization  is  inefficient  as  a  preservative;  the  milk  keeps  only 
twelve  to  twenty-four  hours  longer  than  otherwise.     (This  is  really  no  dis- 
advantage, for  the  quicker  bad  milk  sours,  the  better.) 

/.  Pasteurization  injures  the  taste  of  the  milk.  (This  is  not  so,  if  prop- 
erly done.) 

g.  Pasteurization  increases  the  cost  of  the  milk.  (True,  but  it  is  the 
cheapest  safeguard,  and  the  expense  of  pasteurization  is  offset  by  the  keeping 
quality  of  the  milk.) 

Rosenau  has  made  extensive  tests  to  determine  the  thermal  death -point 
of  those  pathogenic  microbes  most  commonly  found  in  milk.  His  conclu- 
sions are  summarized  as  follows: 

Milk  heated  to  60°  C.  and  maintained  at  that  temperature  for  two  minutes 
will  kill  the  typhoid  bacillus.  The  great  majority  of  these  organisms  are 
killed  by  the  time  the  temperature  reaches  59°  C.,  and  few  survive  to  60°  C. 

The  diphtheria  bacillus  succumbs  at  comparatively  low  temperatures. 
Oftentimes  it  fails  to  grow  after  heating  to  55°  C.  Some  occasionally  sur- 
vive until  the  milk  reaches  60°  C. 

The  cholera  vibrio  is  similar  to  the  diphtheria  bacillus  regarding  its 
thermal  death-point.  It  is  usually  destroyed  when  the  milk  reaches 
55°  C.;  only  once  did  it  survive  to  60°  C.  under  the  conditions  of  the  experi- 
ments. 

The  dysentery  bacillus  is  somewhat  more  resistant  to  heat  than  the 
typhoid  bacillus.  It  sometimes  withstands  heating  at  60°  C.  for  five  minutes. 
All  are  killed  at  60°  C.  for  ten  minutes. 

So  far  as  can  be  judged  from  the  meager  evidence  at  hand,  60°  C.  for 
twenty  minutes  is  more  than  sufficient  to  destroy  the  infective  principle  of 
Malta  fever  in  milk.  M.  melitensis  is  not  killed  at  55°  C.  for  a  short  time; 
the  great  majority  die  at  58°  C.,  and  at  60°  C.  all  are  killed. 

Milk  heated  to  60°  C.  and  maintained  at  that  temperature  for  twenty 
minutes  may,  therefore,  be  considered  safe  so  far  as  conveying  infection 
with  the  micro-organisms  tested  is  concerned. 

Evaporated,  condensed  and  dry  milk  are  found  upon  the  market  and  are 
extensively  used.  Sugar  is  frequently  added  as  a  preservative.  In  making 
condensed  milk,  it  is  evaporated  in  large  pans  until  it  assumes  a  creamy 
consistency.  Dry  milk  is  prepared  by  spraying  the  milk  on  revolving  hot 
cylinders.  The  thin  film  of  milk  is  evaporated  to  dryness  in  a  moment, 


BACTERIA   IN   THE   INDUSTRIES.  1 07 

and  in  that  state  is  scraped  from  the  cylinders.  Dry  milk  is  a  common  in- 
gredient of  baby  foods  and  invalid  foods,  and  is  also  very  extensively  used 
in  the  manufacture  of  chocolate  creams.  The  condensed  and  dry  milks  do 
not  keep  long  in  spite  of  the  greatest  care  in  manufacture.  The  containers 
and  milk  must  be  thoroughly  sterilized  or  pasteurized,  and  the  cans  must 
not  be  opened  until  ready  for  use.  Such  preservatives  as  salicylic  and  boric 
acid  are  sometimes  added  to  condensed  milk. 

It  is  known  that  sweet  cream  yields  a  very  insipid,  flavorless  butter, 
whereas  cream  which  has  " soured"  for  a  few  days  yields  a  pleasant  tasting 
and  pleasingly  flavored  butter,  provided  the  desirable  species  or  variety  of 
bacteria  are  present.  If  the  souring  is  continued  too  long  the  flavor  may 
be  hopelessly  vitiated.  In  the  past  it  was  customary  to  add  a  small  amount 
of  old  cream,  having  a  desirable  flavor,  to  a  new  lot  of  cream.  This  mother 
cream  was  designated  the  "  starter."  It  contained  the  desirable  cream- 
ripening  bacteria,  mostly  of  the  lactic  acid  variety.  These  old-time  natural 
starters  are  now  largely  replaced  by  starters,  prepared  in  the  laboratory 
consisting  of  pure  cultures  of  certain  breeds  or  varieties  of  cream  flavor, 
producing  germs  of  the  lactic  acid  group.  A  proper  regulation  of  the  tem- 
perature is  very  important  in  the  ripening  of  cream  (60°  to  75°  F.).  It  is 
also  necessary  to  pasteurize  the  cream  before  adding  the  bacterial  starter 
in  order  to  prevent  the  development  of  microbes  which  might  interfere  with 
the  proper  development  of  the  starter  microbes.  Naturally  the  use  of  clean, 
sterilized  utensils  and  uniformity  of  methods  are  all-important,  in  order  that 
uniform  results  may  be  obtained. 

Cheese  flavors  are  also  due  to  bacterial  action,  but  not  wholly  so, 
as  many  of  the  higher  fungi,  as  species  of  Penicillium  (Camembert 
Penicillium)  and  of  Oidium  (O.  lactis)  also  play  a  very  important  part  as 
flavor  producers.  The  Roquefort  cheese  owes  its  characteristic  flavor,  in 
part  at  least,  to  a  variety  or  form  of  Penicillium  glaucum.  The  qualities  and 
properties  of  some  Swiss  and  soft  Belgian  cheeses  are  largely  due  to  Oidium 
lactis.  The  ripening  of  hard  cheeses  (Cheddar,  Edam,  American,  some 
Swiss  varieties,  and  others)  is  due  exclusively  to  bacterial  action.  Cream, 
butter  and  cheese  are  very  prone  to  the  attacks  of  objectionable  bacteria  and 
moulds  which  cause  very  unpleasant  flavors  and  bitter  taste. 

It  must  also  be  borne  in  mind  that  cream,  cheese  and  butter  may  carry 
disease  germs.  Tubercle  bacilli  have  been  reported  in  these  food  articles, 
but  it  has  not  been  demonstrated  that  they  are  frequently  present.  Typhoid 
infections  have  been  traced  to  the  use  of  cream,  but  no  case  of  typhoid  fever 
has  ever  been  definitely  traced  to  eating  butter  or  cheese.  Of  course,  these 
articles  may  become  infected  after  manufacture  and  thus  become  a  possible 
means  of  spreading  disease. 


108  PHARMACEUTICAL   BACTERIOLOGY. 

3.  The  Lactic  Acid  Microbe  and  Kefir  Preparation. 

Within  recent  years  the  subject  of  intestinal  digestion  and  the  relation- 
ship of  intestinal  microbes  to  digestion  and  longevity  has  received  much 
attention.  Metchnikoff  declares  that  the  early  senile  cell  changes  in  the 
body  are  due  to  the  repeated  or  chronic  autointoxications  brought  about 
by  certain  noxious  intestinal  ferments  of  bacterial  origin  which  are  absorbed 
into  the  circulation.  Some  of  these  bacteria,  especially  those  found  in  the 
small  intestines,  are  beneficial,  secreting  enzymes  which  aid  digestion,  but 
the  enormous  quantity  of  microbes  active  in  the  lower  large  intestine  are  for 
the  most  part  injurious,  producing  putrefactive  changes,  liberating  toxins 
which  when  absorbed  into  the  system  in  sufficient  quantity  produce  the 
symptoms  of  ptomaine  poisoning. 

In  order  to  combat  these  objectionable  bacterial  activities,  it  is  necessary 
to  regulate  the  bacterial  development  in  the  large  intestine.  Lactic  acid  has 
long  been  known  as  an  efficient  remedy  in  the  treatment  of  various  intestinal 
disorders.  It  is  known  that  the  poor  of  certain  European  countries  who 
live  largely  on  potatoes  and  clabbered  or  thick  milk  are  notably  free  from 
intestinal  disorders  and  are  remarkably  long-lived.  It  is  known  that  pickles, 
sauerkraut  and  sour  milk  are  excellent  bowel  regulators,  in  spite  of  the  fact 
that  these  foods,  the  former  two  in  particular,  are  well-nigh  indigestible  and 
have  little  food  value.  The  Arabians  have  long  used  koumys  as  a  healthful, 
life-prolonging  article  of  diet.  To  this  class  of  foods  also  belongs  the  Bul- 
garian yoghurt  and  the  Egyptian  raib. 

The  ferments  of  koumys,  kefir,  yoghurt  and  raib  resemble  each  other  in 
that  they  are  mixed,  consisting  of  several  lactic-acid  microbes  or  organisms 
and  yeast  organisms.  These  foods  or  drinks  therefore  contain  lactic  acid 
and  a  small  amount  of  alcohol. 

As  soon  as  it  was  determined  experimentally  that  the  beneficent  action  of 
sour  milk,  thick  or  clabbered  milk  and  the  above-named  special  preparations 
was  largely  due  to  the  lactic  acid  formed  by  specific  microbes,  efforts  were 
made  to  isolate  these  organisms  in  pure  culture  and  to  induce  them  to  act 
in  sterile  or  pure  milk.  This  has  been  done,  and  there  are  now  upon  the 
European  and  American  market  several  patented  preparations  consisting 
of  the  lactic  acid  bacillus. 

Our  knowledge  of  the  relative  importance  of  the  several  organisms  which 
are  said  to  produce  the  fermentative  changes  in  the  milk  is  as  yet  incom- 
plete. Bacteriologists  have  thus  far  not  succeeded  in  disclosing  all  of 
nature's  secret  processes  involved.  It  is  supposed  that  the  microbe  of 
Bulgarian  sour  milk,  the  Bacillus  bulgaricus,  is  the  most  vigorous  and  active 
of  all  organisms  concerned  in  the  lactic-acid  fermentation  of  milk. 

It  is  not  definitely  determined  whether  or  not  the  fermentations  of  milk 


BACTERIA   IN    THE   INDUSTRIES.  109 

induced  by  the  mixed  and  often  filthy  "yeasts"  employed  in  making  koumys, 
kefir,  yoghurt,  matzoon  and  other  similar  fermented  milk  foods,  are  superior 
or  inferior  to  those  of  lactone  and  other  pure  culture  milk  ferments^  It  is, 
however,  very  evident  that  the  marketed  preparations  in  tablet  form  give 
very  satisfactory  results,  as  used  by  pharmacists  and  in  the  home.  Full 
directions  for  using  the  tablets  are  found  on  every  package.  As  is  natu- 
rally to  be  supposed,  these  tablets  deteriorate  in  a  comparatively  short  time 
and  all  reliable  manufacturers  place  the  age-limit  on  each  package. 

Pharmacists  can  prepare  a  marketable  kefir  ferment  powder  from  milk 
activated  by  kefir,  provided  care  is  observed  to  guard  against  outside  infec- 
tion in  the  several  steps  of  procedure.  The  following  is  the  method  of  pre- 
paring a  kefir  powder: 

A.  Securing  the  Kefir. — The  kefir  known  as  kefir  grains  or  kefir  seeds 
may  be  secured  from  the  large  dealers  in  drugs  in  New  York  City  or  in 
other  large  Eastern  port  cities.     The  kefir  is  a  solid  of  a  tough  gelatinous 
consistency,  brittle  when  dry,  of  grayish-yellow  color.     It  is  a  conglomera- 
tion of  various  organisms,  as    Dispora  caucasica,  several  species  of  other 
microbes,  a  yeast  organism,  and  other  undetermined  organisms. 

B.  Washing  the  Kefir. — Place  two  or  three  drams  of  the  kefir  in  a  mixture 
of  equal  parts  of  milk  and  water,  enough  to  cover  the  kefir.     Allow  to  stand 
for  four  hours,  decant  off  the  liquid  and  renew  at  intervals  of  about  one  hour. 
Repeat  this  four  or  five  times  at  a  temperature  of  about  82°  F. 

This  process  serves  a  cleansing  purpose  and  initiates  the  fermentative 
change.  The  amount  used  will  depend  upon  the  quantity  of  powder  to  be 
made. 

C.  Preparing  the  New  Kefir. — Wrap  the  washed  and  softened  kefir  in  a 
piece  of  sterilized  gauze  and  place  it  in  one  quart  of  pasteurized  milk.     Keep 
at  a  temperature  of  82°  F.     Allow  to  stand  for  from  twelve  to  fifteen  hours, 
until  the  milk  is  curdled. 

D.  Skimming  and  Draining  the  Kefir. — Remove  the  cream  and  drain  the 
curd  (kefir)  in  sterilized  gauze  until  quite  dry. 

E.  Drying. — Add  (to  the  drained  kefirized  curd)  an  equal  weight  of 
sugar  of  milk,  mix,  and  spread  thinly  upon  sterilized  gauze  or  upon  a  sterile 
glass  plate  and  dry  in  a  current  of  sterile  warm  air  (80°  F.) 

F.  Powdering. — Powder  the  dried  mass  gently  and  put  up  in  dry,  sterile, 
one-ounce,  wide-mouthed  vials,  closed  with  sterilized  corks. 

G.  Directions  for  Use. — Upon  the  bottles  place  the  following  directions 
for  using  the  powder  thus  prepared:     "Dilute  one  quart  of  milk  with  one- 
half  pint  of  water,  add  a  pinch  of  salt  and  one  level  teaspoonful  of  the  powder. 
Set  aside  for  twelve  to  fifteen  hours  at  a  temperature  of  85°  F.,  shaking 
frequently.     Use  at  once  or  keep  on  ice." 

There  are,  of  course,  no  conveniences  for  regulating  the  temperature  in 


IIO  PHARMACEUTICAL   BACTERIOLOGY. 

the  average  household,  and  the  action  of  the  powder  must  take  place  at  the 
ordinary  temperature  of  the  home.  Thus  the  time  required  to  curdle  the 
milk  will  vary.  The  powder  should  be  kept  in  a  cool  or  cold,  dry  place. 
Of  course,  a  small  amount  of  kefirized  milk  can  be  used  to  curdle  any  quan- 
tity of  fresh  milk  without  using  any  of  the  powder. 

The  pharmacist  should  test  the  kefir  which  he  is  about  to  use  in  preparing 
the  powder,  in  order  to  be  certain  that  it  is  active  in  curdling  milk.  Like- 
wise should  he  test  the  powder  prepared  from  it. 

The  kefir  powder  above  described  is  similar  to,  although  not  identical 
with,  certain  microbic  lactic-acid  ferments  found  on  the  market,  as  the 
lactone  tablets,  bacillary  tablets,  yoghurt  tablets,  fermenlactyl,  lacto-bacilline 
and  others.  These  are  prepared  from  pure  cultures  of  species  of  lactic-acid 
bacilli,  dried  and  formed  into  tablets  with  some  pulverulent  (starch,  milk, 
sugar)  base,  ready  for  use.  The  milk  (in  quart  bottles)  is  first  pasteurized, 
a  pinch  of  salt  is  added  and  two  or  three  tablets  are  crushed  and  mixed 
with  the  milk.  In  a  day  or  so  the  milk  is  transformed  into  an  acidulous 
drink,  resembling  buttermilk  somewhat  in  flavor,  though  it  is  not  buttermilk, 
as  is  generally  supposed. 

These  tablets  have  gained  in  favor  within  recent  years.  They  deteriorate 
in  time,  as  already  stated,  and  the  time-limit  is  stamped  on  each  container. 
Like  the  kefir,  they  act  more  quickly  at  a  temperature  of  about  25°  C. 

As  may  be  readily  understood,  kefir,  lactone,  etc.,  will  not  produce  the 
characteristic  changes  in  milk  to  which  preservatives  have  been  added;  in 
fact,  the  failure  to  produce  fermentation  is  an  indication  that  preservatives 
are  present. 

4.  Microbic  Pest  Exterminators. 

Attempts  have  been  made  from  time  to  time  to  exterminate  certain  animal 
pests  by  inoculating  them  with  some  fatal  contagious  disease  of  microbic 
origin.  Experiments  along  this  line  have  been  carried  on  for  some  time, 
ever  since  the  causative  relationship  of  microbes  and  disease  was  fully  estab- 
lished; but  it  is  only  within  recent  years  that  extensive  practical  application 
was  made  of  the  use  of  a  few  microbic  pest  exterminators.  One  of  the  first 
to  be  used  with  some  success  was  the  chintz-bug  exterminator.  The  chintz- 
bug  (Blissus  leucopteris,  also  called  chinch-bug,  chink-bug)  was  a  very 
destructive  corn  (Zea  mays}  pest  of  the  Central  States  (Illinois,  Kansas, 
Nebraska,  Iowa),  causing  great  damage  to  crops  during  certain  very  dry 
seasons.  Extensive  experiments  carried  on  at  the  University  of  Illinois  and 
also  at  the  University  of  Minnesota  (Departments  of  Agriculture)  led  to  the 
discovery  of  a  microbic  disease  of  this  pest  which  was  quickly  fatal  and  which 
spread  very  rapidly.  The  insects,  in  cages,  were  inoculated  with  pure 
cultures  of  the  pathogenic  microbes,  and  insects  in  the  diseased  condition 


BACTERIA   IN   THE   INDUSTRIES.  Ill 

were  sent  to  the  farmers  with  instructions  how  to  scatter  them  through  an 
infested  corn-field.  The  results  were  in  some  instances  very  satisfactory, 
and  again  without  appreciable  effects.  The  trouble  in  the  use  ofjhis  exter- 
minator lay  in  the  fact  that  the  climatic  conditions  (rainy,  damp  weather) 
essential  to  the  spreading  of  the  disease  did  not  generally  prevail,  and  as 
soon  as  the  climatic  conditions  were  favorable  inoculation  became  unneces- 
sary, as  the  disease  developed  without  artificial  aid  and  effectually  checked 
further  ravages. 

Rabbits  are  one  of  the  very  annoying  field  pests  of  Australia,  and  at- 
tempts have  been  made  to  exterminate  them  by  means  of  pure  cultures  of 
microbes  capable  of  developing  a  fatal  infectious  disease  among  these 
animals,  but  the  results  were  quite  unsatisfactory. 

More  recently  there  have  been  placed  on  the  market  quite  an  array  of 
mice  and  rat  exterminators  of  microbic  origin  under  various  trade  names 
as  ratin,  rat  virus,  azoa,  rattite,  Danysz  virus  and  mouratus.  These  prep- 
arations consist  of  pure  cultures  of  bacilli  pathogenic  to  rats  and  mice,  as 
the  Bacillus  murisepticus  and  Bacillus  ty'phimurium,  mixed  with  some  inert 
base,  as  corn-meal,  oat-meal,  etc.,  forming  a  coarse  powder.  Some  prep- 
arations are  in  liquid  form.  They  are  used  by  mixing  the  powder  or  liquid 
with  moist  corn-meal  or  other  food  material  relished  by  these  animals,  and 
spreading  it  near  their  haunts  and  runs.  Fortunately,  these  substances  are 
harmless  to  man  and  animals  other  than  mice  and  rats.  These  microbic 
rat  and  mice  exterminators  have  thus  far  proven  to  be  rather  unsatisfactory. 
They  have  undoubtedly  given  excellent  results  in  some  instances,  and  again 
they  have  been  absolute  failures.  The  tests  made  by  the  University  of 
California,  and  by  Dr.  Rupert  Blue  in  his  famous  plague-rat  extermination 
in  San  Francisco,  have  given  almost  wholly  negative  results.  A  microbic 
squirrel  exterminator  ("squirrelin")  has  proven  entirely  unsatisfactory. 

When  we  consider  how  difficult  it  is  to  prevent  fatal  epidemics,  it  cer- 
tainly does  seem  reasonable  to  suppose  that  it  should  be  a  comparatively  easy 
matter  to  find  ways  and  means  for  disseminating  fatal  epidemics,  but  so  far 
the  commercial  attempts  made  in  that  direction  have  proven  rather  discourag- 
ing. Further  carefully  conducted  experiments  along  this  line  are  necessary. 
It  is  known '  that  the  ravages  of  certain  pests  are  sometimes  suddenly 
checked  by  the  natural  invasion  of  some  pathogenic  organisms.  This  is  fre- 
quently observed  among  plant  lice  (Aphis)  and  other  insect  enemies  of  plants. 

5.  Bacteria  in  the  Tanning  Industry. 

The  object  in  tanning  leather  is  to  protect  it  against  decomposition  and 
to  render  it  pliable.  The  various  animal  hides  before  reaching  the  tannery 
are  preserved  by  drying  and  salting.  At  the  tannery  the  hides  are  treated 
as  follows: 


112  PHARMACEUTICAL   BACTERIOLOGY. 

A.  Removing  the  Hair — Depilation. — This  is  done  by  means  of  chemicals, 
as  lime  or  sodium  sulphite,  or  through  the  agency  of  rotting  bacteria,  as 
Bacillus  vulgaris   (Proteus")    and   others.     Just  which   of   several    species 
of  rotting  bacteria  is  most  active  in  this  process  has  not  been  definitely 
determined. 

B.  Drenching  or  Bating. — After  the  hair  has  been  removed,  the  hides 
are  macerated  in  an  aqueous  solution  of  the  excrement  or  dung  of  pigeons, 
hens  and   dogs.     These  substances  set  up  a  lactic  acid  fermentation  due  to 
the  microbes  contained  therein.     The  active  organisms  have  not  been  iso- 
lated as  yet;  Bacillus  gasoformans  and  B.  erodiens  are  perhaps  active,  but  there 
are  also  present  many  yeasts,  moulds  and  other  organisms  which  may  have 
their  special  effects. 

The  first  part  of  this  process,  known  as  "  bating,"  is  initiated  by  bird 
dung;  the  second  process,  known  as  "puring,"  is  due  to  the  action  of  dog 
dung.  Attempts  have  been  made  to  use  pure  cultures  of  the  active  microbes 
to  supplant  these  filth  substances,  but  so  far  these  efforts  have  not  proven 
wholly  successful. 

C.  Tanning. — The  bated  hides  are  next  treated  in  the  tan  pit  (coarse 
skins)  or  in  bark  liquor  (soft  thin  skins),  where  the  souring  process  takes 
place.     This  process  is  also  due  to  bacterial  activity.     Our  knowledge  of 
the  action  which  takes  place  and  of  the  bacteria  involved  is  very  incomplete. 

Bacteria  are  important  factors  in  siloing;  in  curing  tobacco,  tea  and 
cacao.  The  flavor  of  different  brands  of  tobacco  is  due  to  different  bacteria, 
and  attempts  have  been  made  to  isolate  those  producing  desirable  flavors 
and  to  use  them  in  pure  culture.  It  is  highly  probable  that  the  bouquet  of 
old  wines  is  due  to  bacterial  action.  These  are,  however,  matters  which 
require  further  study.  Rotting  bacteria  are  active  in  paper-making.  In 
the  maceration  process  certain  bacteria  feed  upon  and  decompose  the  less 
resisting  vegetable  cell-walls,  as  those  of  the  parenchymatous  tissues,  the 
epidermal  tissue,  etc.,  leaving  the  more  resisting  fibrous  lignified  tissues  as 
bast  and  wood  fibers.  The  pulp  is  then  poured  on  sieves  and  the  rotted 
or  digested  portions  washed  out. 

Bacteria  are  now  practically  employed  in  the  -  purification  of  sewage. 
This  is  done  in  what  are  known  as  "contact  beds,"  in  which  the  environ- 
ment is  made  favorable  to  rapid  development  of  those  non-pathogenic  rotting 
bacteria  which  disintegrate  the  organic  substances  and  at  the  same  time 
prevent  the  development  of  the  pathogenic  or  otherwise  objectionable  mi- 
crobes. It  is  highly  probable  that  this  method  may  be  applied  to  the  puri- 
fication of  streams  and  other  large  bodies  of  water. 

The  possibilities  in  the  practical  utilization  of  bacteria  in  the  arts  and 
industries  are  promising,  and  it  may  confidently  be  expected  that  wonderful 
innovations  along  this  line  will  be  made  in  the  very  near  future. 


BACTERIA  IN   THE   INDUSTRIES.  113 

6.  Cider-making. 

Acetic-acid  fermentation  in  wine  cider  and  other  fermented  alcoholic 
substances  is  initiated  by  the  Mycoderma  aceti,  collectively  known  as  "mother 
of  vinegar."  This  is  no  doubt  a  mixed  growth,  representing  several  species 
or  varieties  of  acetic-acid  forming  organisms.  While  it  is  true  that  nature 
invariably  inoculates  the  substances  named,  resulting  in  the  production  of 
vinegar,  it  is  customary  to  use  the  top  skin  or  pellicle  (mother  of  vinegar)  on 
vinegar  already  formed,  adding  it  to  new  wine  or  cider  in  order  to  hasten  the 
fermentation.  As  stated,  this  is  not  a  pure  culture  representing  a  single 
species.  In  fact,  the  tests  with  what  were  pure  species  have  proven  un- 
satisfactory. The  vinegar  organisms  require  an  abundance  of  oxygen.  To 
supply  the  necessary  oxygen  (of  the  air)  it  is  customary  to  have  the  fermen- 
tation barrels  or  casks  only  about  two-thirds  or  three-fourths  full  and  to 
leave  the  bunghole  open  (generally  with  a  plug  of  cotton) .  In  Germany  a 
quickened  method  is  much  in  vogue.  The  wine  or  cider  is  allowed  to  trickle 
slowly  through  a  cask  filled  with  wood  shavings  which  are  moistened  with 
old  vinegar.  The  wood  shavings  offer  a  maximum  surface  exposure  and 
fermentation  is  as  a  result  very  much  hastened. 

Occasionally  the  vinegar  loses  its  acidity.  This  is  due  to  the  invasion  of 
a  bacillus  (B.  xylenum)  which,  in  the  presence  of  oxygen,  splits  up  the  acetic 
acid  into  other  compounds.  This  change  can  be  prevented  by  excluding 
air  from  the  containers.  Vinegar  should  contain  from  4  to  4 . 5  per  cent,  of 
acetic  acid  (the  legal  standard) . 


CHAPTER  VII. 


IMMUNITY  AND  IMMUNIZING  AGENTS. 

We  speak  of  immunity  from  and  susceptibility  to  disease,  indicating 
thereby  a  difference  in  individuals  regarding  their  responsive  behavior  to  the 
factors,  forces  or  influences  which  may  cause  or  prevent  disease  manifesta- 
tions. The  subject  is  one  of  intense  interest  in  the  light  of  modern  biological 
and  bacteriological  investigations.  It  has  within  recent  years  received  more 
attention  from  biologists,  physiologists  and  bacteriologists  than  any  other 
branch  of  science  and  some  of  the  results  obtained  are  in  many  respects 
marvelous.  The  discoveries  thus  far  made  are  the  mere  beginnings  of  future 
preventive  medicine,  which  will  make  it  possible  to  establish  a  system  of 
medical  practice  of  which  the  chief  aim  will  be  to  prevent  rather  than  to 
cure  disease. 

Several  kinds  of  immunity  are  recognized  which  may  be  tabulated  as 
follows: 


Immunity 


Inherited 


Racial        ] 
(Phylogenetic)  j 


As  observed  in  the  different  orders, 
families,  genera  and  species  of  the 
animal  kingdom. 


Individual    ' 


Induced 


Active 


As  observed  in  different  individuals  of  the 
same  species  or  variety.    (Ontogenetic.) 


As  observed  in  the  sexes  of  the  same 
species  or  variety. 


'  Due  to  naturally  induced 
infections  with  disease 
which  produce  immunity 
to  subsequent  attacks, 
as  diseases  of  childhood, 
acclimatization,  etc. 


Natural 


Artificial 


Due  to  use  of  modified 
toxins,  bacterins,  and 
direct  inoculation  with 
disease  germs. 


Passive — Use  of  antitoxins  and  other  disease  preventives. 
114 


IMMUNITY  AND   IMMUNIZING  AGENTS.  115 

It  has  been  known  for  a  long  time  that  when  a  number  of  individuals  of 
the  same  species  are  exposed  to  the  same  bacterial  infection,  some  escape  the 
infection  while  others  do  not.  That  which  prevented  the  development  of  the 
bacterial  disease  or  which  neutralized  the  toxic  products  or  which  killed  the 
disease  organisms  thus  preventing  the  disease  manifestations,  constitutes 
immunity. 

It  may  be  assumed  that  the  members  of  the  various  subdivisions  of  the 
animal  kingdom  have  in  the  course  of  their  phylogenetic  or  evolutional  de- 
velopment acquired  certain  properties  of  cells,  cell-contents,  tissues  and 
organs,  which  enable  them  to  resist  certain  harmful  bacterial  invasions,  as 
well  as  the  injurious  effects  of  other  noxious  influences  and  substances. 
For  example,  the  typhoid  fever  germ  is  harmless  to  the  oyster  and  other  lower 
as  well  as  most  higher  animals,  but  it  is  very  injurious  to  man.  The  vege- 
table alkaloids  are  very  toxic  to  man  and  most  other  vertebrates,  whereas 
they  are  harmless  to  the  protozoa  and  other  low  forms  of  animal  life.  As  is 
known  drug  parasites  feed  with  impunity  upon  the  most  potent  vegetable 
drugs.  The  carnivora  are  less  liable  to  bacterial  infection  than  the  her- 
bivora.  Closely  related  species  sometimes  display  remarkable  immunity 
differences.  For  example,  field  mice  are  very  susceptible  to  glanders,  whereas 
the  common  house  mouse  is  almost  wholly  immune.  Jersey  cows  are  less 
liable  to  tuberculosis  than  Holsteins.  The  Yorkshire  breed  of  swine  is  less 
liable  to  the  attacks  of  hog  erysipelas  than  are  other  breeds.  Man  is 
especially  susceptible  to  malaria,  cholera  and  typhoid  fever.  Man,  cattle 
and  apes  are  very  susceptible  to  infection  by  the  tubercle  bacillus,  whereas 
the  wild  carnivora  are  quite  exempt.  Anthrax  may  attack  man,  cattle, 
sheep  and  guinea-pigs,  whereas  birds,  rats,  cats  and  dogs  are  free  from  such 
attack.  The  Caucasian  race  is  less  liable  to  small-pox,  tuberculosis  and 
syphilis  than  is  the  Negro  race.  On  the  other  hand,  the  Negro  is  more 
immune  to  yellow  fever  than  is  the  white  man. 

Immunity  is,  however,  very  largely  relative.  The  wild  carnivora  are  quite 
free  from  disease  whereas  in  prolonged  captivity  they  may  fall  prey  to 
several  diseases,  notably  tuberculosis.  Toxic  substances,  noxious  gases, 
lack  of  food,  poor  food,  cold,  excessive  heat,  fatigue,  over-exertion,  inclement 
weather,  etc.,  are  factors  which  may  lessen  the  natural  immunity  to  the 
several  infections  to  which  the  animal  may  be  exposed.  For  example,  no 
race  of  mankind  is  possessed  of  absolute  immunity  to  any  human  disease. 
Such  immunity  differences  as  are  observed  are  due  to  differences  in  the  op- 
portunities for  infection,  differences  in  habit,  in  occupation,  etc. 

The  modern  explanations  regarding  the  mechanism  of  immunity  are 
extremely  interesting  and  a  work  on  pharmaceutical  bacteriology  would 
certainly  be  incomplete  without  a  brief  summary  of  the  discoveries 
to  date. 


Il6  PHARMACEUTICAL  BACTERIOLOGY. 

In  1890  Behring  and  Kitasato  found  that  the  cell-free  blood  (serum)  of 
rabbits  and  of  mice  which  had  been  artificially  immunized  against  tetanus, 
neutralized  or  destroyed  the  toxic  substances  of  the  tetanus  bacillus.  To 
this  substance  they  gave  the  name  antitoxin.  This  was  an  epoch-making 
discovery.  It  led  to  the  finding  of  other  antitoxins  or  antibodies  which  are 
now  used  in  the  treatment  of  disease  as  will  be  more  fully  explained  in  a  sub- 
sequent chapter.  Antitoxins,  like  the  toxins,  possess  many  of  the  characters 
of  albuminoids,  are  quite  readily  decomposed  and  are  incapable  of  isolation 
from  the  blood  or  from  the  tissue  cells.  Never  having  been  obtained  in 
purity  nothing  is  known  regarding  their  physical  appearance.  They  are 
readily  destroyed  at  comparatively  low  temperatures  (65°  to  75°  C.)  and  by 
exposure  to  light  and  air.  They  are  very  sensitive  to  acids  and  are  best  pre- 
served by  evaporating  the  blood  sera  in  which  they  are  contained  to  dryness 
in  a  vacuum  at  a  low  temperature  and  storing  in  a  vacuum,  at  a  low  tempera- 
ture, away  from  light  and  in  a  dry  place.  Experimentally  it  has  been  dem- 
onstrated that  the  antitoxins  are  intimately  combined  with  the  globulins 
of  the  blood.  This  discovery  led  to  the  manufacture  of  concentrated  anti- 
toxins by  precipitating  the  globulins  with  ammonium  sulphate,  magnesium 
sulphate  and  other  salts.  Remarkably  enough,  reactions  have  been  ob- 
served which  would  indicate  that  antitoxin  is  not  a  proteid  substance;  for 
example,  it  is  not  destroyed  (digested)  by  trypsin. 

It  has  furthermore  been  found  that  variably  small  amounts  of  antitoxins 
exist  in  normal  blood;  that  is,  in  the  blood  of  animals  that  have  not  been 
naturally  or  artificially  immunized,  and  also  in  still  lesser  amounts  in  the  milk 
of  normal  animals.  As  to  the  origin  of  the  antitoxins  the  physiologic  evi- 
dence points  to  their  formation  in  the  body  cells  rather  than  in  the  blood 
serum. 

Another  important  discovery  was  that  normal  blood  could  actively  de- 
stroy (lake)  bacteria,  and  in  common  with  antitoxins  this  bactericidal  prop- 
erty was  found  to  be  specific.  That  is,  serum  found  to  be  quite  destructive 
to  the  typhoid  bacillus  is  not  destructive  to  the  cholera  bacillus.  These 
germ  destroying  or  bactericidal  substances  are  designated  lysins.  Ehrlich 
has  discovered  that  there  are  in  fact  three  distinct  blood  lysins;  namely, 
cytolysin,  a  substance  which  is  capable  of  destroying  (laking)  body  cells; 
hemolysin,  which  is  capable  of  destroying  red  blood-corpuscles;  and  bactero- 
lysin  as  already  explained.  By  injecting  tissue  cells,  as  those  of  kidney  or 
of  some  other  organ,  into  an  animal,  there  are  developed  in  the  blood  of  the 
inoculated  animal  lysins  which  will  dissolve  kidney  cells  or  other  organ  cells 
used.  If  the  blood  of  a  bird  or  other  animal  is  injected  into  an  animal  of  a 
different  species,  hemolysins  will  appear  in  the  blood  of  the  animal  thus 
injected.  This  hemolysin  is  specific,  as  it  will  only  dissolve  or  destroy  the 
hemoglobin  in  the  blood  of  the  kind  of  animal  of  which  the  blood  was  used 


IMMUNITY  AND  IMMUNIZING  AGENTS.  1 17 

for  injecting.  An  animal  inoculated  with  the  typhoid  bacillus  will  produce 
a  blood  lysin  which  destroys  the  typhoid  bacillus.  Lytic  sera  become  in- 
active when  heated  to  55°  C.  for  one-half  hour  and  such  sera  are  said  to  be 
inactivated.  However,  if  normal  serum  is  added  to  the  inactivated  serum 
the  bactericidal  power  is  fully  restored.  The  bactericidal  power  of  the 
serum  can  be  greatly  increased  by  the  use  of  highly  virulent  bacterial  cultures, 
thus  producing  a  serum  of  high  potency.  In  actual  practice,  as  in  the  man- 
ufacture of  bactericidal  sera  for  the  prevention  and  cure  of  disease,  the  ani- 
mal (as  horse)  is  first  inoculated  with  attenuated  cultures,  then  with  normally 
virulent  cultures  and  finally  with  hyper-virulent  cultures  of  the  specific 
pathogenic  microbe.  Such  sera  act  by  destroying  the  disease-producing 
bacteria,  but  they  have  no  effect  upon  the  toxins  produced  by  the  bacteria, 
thus  showing  that  they  are  entirely  distinct  from  the  antitoxins. 

The  eminent  bacteriologist  Metchnikoff  made  the  very  interesting 
discovery  that  the  white  blood-corpuscles  (leucocytes)  had  the  power  of 
feeding  upon  and  digesting  bacteria  with  which  they  came  in  contact.  That 
is  the  white  blood-corpuscles,  called  phagocytes,  act  as  the  defenders  of  the 
body  against  bacterial  invasion.  This  observation  by  Metchnikoff,  fully 
verified  by  others,  is  generally  known  as  the  phagocyte  theory  and  the 
phenomenon  is  designated  phagocytosis.  The  principle  involved  in  phago- 
cytic  activity  is  well  illustrated  in  the  lesser  local  injuries,  as  cuts,  bruises, 
abrasions,  etc.  Normally  such  injuries  are  always  infected  by  various 
germs  of  the  environment,  as  the  several  varieties  of  pus  microbes.  These 
invading  microbes  at  once  begin  their  attack  upon  the  tissue  cells  and 
blood-corpuscles.  The  leucocytes  which  are  present  begin  to  feed  upon 
the  rapidly  multiplying  pus  organisms  but  for  a  time,  as  a  rule,  the  latter 
have  the  upper  hand  and  as  a  result  there  is  perceptible  pus  formation 
("the  laudable  pus"  of  older  writers)  represented  by  dead  leucocytes  gorged 
with  microbes.  As  the  inflammatory  reaction  becomes  more  marked,  in- 
dicated by  redness  and  swelling  of  the  tissues  immediately  about  the  injury; 
increased  numbers  of  leucocytes  (phagocytes)  are  brought  to  the  scene  of 
action  and  gradually  they  gain  control  until  finally  the  invading  microbes  are 
all  destroyed,  thus  permitting  a  rapid  and  unhindered  restoring  of  tissue  cells, 
recognized  as  the  healing  process.  This  phagocytic  action  is  entirely 
distinct  from  the  action  of  antitoxins  and  lysins,  and  the  three  are  potent 
factors  in  immunity. 

The  investigations  of  Metchnikoff  and  Leishman  on  phagocytosis  paved 
the  way  for  the  discovery  of  opsonins  by  Wright.  It  was  noticed  that  the 
phagocytic  activity  was  influenced  by  conditions  to  be  found  outside  of  the 
leucocytes  themselves.  Metchnikoff  held  that  the  principal  part  is  played 
by  substances  found  in  the  serum  and  in  the  tissue  cells  to  which  he  gave  the 
name  "stimulins."  The  purpose  of  these  substances  in  the  tissue  fluids 


Il8  PHARMACEUTICAL    BACTERIOLOGY. 

have  not  yet  been  satisfactorily  demonstrated,  but  Metchnikoff  considers 
their  function  to  be  that  of  acting  upon  the  phagocytes  in  such  a  manner  as  to 
stimulate  them  to  perform  phagocytosis.  Wright,  Hektoen,  Neufeld  and 
others  have  demonstrated  beyond  doubt,  the  presence  in  the  blood  of 
substances  which  act  upon  the  infecting  bacteria  and  get  them  ready  fpr 
the  completion  of  their  destruction  by  the  phagocytes.  To  these  bodies 
he  has  given  the  name  "opsonins"  (Latin,  opsono,  I  prepare  for).  That 
opsonins  are  not  formed  in  the  blood  is  certain.  Experimental  evidence 
seems  to  prove  that  they  are  products  of  muscular  or  subcutaneous  cellular 
activity.  It  is  probable  that  the  actual  formation  of  opsonin  occurs  in 
the  muscle  tissues  and  passes  thence  to  the  blood.  Wright  has  demon- 
strated more  or  less  satisfactorily  the  presence  of  opsonins  in  the  blood 
of  animals  and  humans  and  by  a  special  technic  has  measured  the  relative 
amount.  This  measurement  is  a  ratio  of  the  activity  of  the  phagocytes  in 
normal  blood  and  of  that  in  disease,  before  and  after  stimulation,  determined 
by  the  number  of  bacteria  that  a  single  phagocyte  will  ingest — the  so- 
called  opsonic  index.  This  index  or  ratio  is  made  intelligible  by  decimal 
figures  representing  the  number  of  bacteria  which  the  average  phagocyte 
will  take  up.  We  may  assume  that  one  phagocyte  in  normal  blood  will 
ingest  an  average  of  10  bacteria,  represented  in  the  index  by  the  figures  i.o, 
but  in  disease  (chronic)  the  phagocytes  may  only  take  up  an  average  of  3,  6, 
or  other  numbers,  represented  by  the  figures  0.3,  0.6,  etc.  After  stimulation 
the  phagocytes  may  take  up  15,  25,  or  even  numbers  of  bacteria  represented 
in  the  index  by  the  figures  1.5,  2.5,  etc. 

Taking  the  opsonic  index  of  an  individual's  blood  calls  for  considerable 
delicate  technic.  In  brief,  it  is  performed  by  mixing  together  equal  vol- 
ume quantities  (measured  in  a  capillary  tube)  of  blood  serum  and  an  emulsion 
of  bacteria  and  incubating  for  15  minutes  at  37.5°  C.  Then  making  a  thin 
smear  of  the  mixture  on  a  microscope  slide,  drying  and  staining,  and  count- 
ing the  number  of  bacteria  enclosed  in  each  white  blood-corpuscle  (50  to  200 
cells  counted)  and  striking  an  average.  This  average  is  the  index  stated 
by  a  decimal  figure.  The  index  thus  obtained  indicates  the  relative 
phagocytic  power  of  the  individual's  blood  tested,  whether  below  or  above 
the  normal. 

The  opsonic  index  taken  in  the  various  chronic  forms  of  bacterial  infec- 
tions is  invariably  below  normal  and  shows  that  the  phagocytic  power  is 
low,  and  it  seems  to  prove  that  the  chronicity  is  due  to  the  abnormal  phago- 
cytosis. The  injection  of  several  millions  of  devitalized  bacteria  of  the 
kind  causing  the  infection,  induces  the  formation  of  the  specific  opsonin, 
arouses  the  phagocytic  activity  and  corrects  the  pathologic  condition. 
The  opsonic  method  of  treatment  has  been  extensively  tested  through 
the  use  of  specifically  active  bacterial  suspensions  (vaccines,  bacterins  or 


IMMUNITY  AND   IMMUNIZING  AGENTS. 


119 


opsonogens)  which  in  some  instances  have  given  excellent  results.  It  has 
also  been  found  that  substances  other  than  opsonins  may  increase  phagocy- 
tosis, as  for  example,  nucleinic  acid  and  collargol. 

From  the  foregoing  it  becomes  evident  that  immunity  from  disease 
depends  upon  the  presence  in  the  body  of  antitoxins,  bacterolysins,  and  the 


FIG.  53. — Opsonic  Incubator.  The  determination  of  the  Opsonic  Index  has  become 
so  important  that  these  incubators  have  been  made  to  meet  the  demands  for  an 
apparatus  in  which  twenty  pipettes  can  be  incubated  at  one  time,  and  so  that  any  tube  may 
be  examined  during  the  progress  of  the  experiment  without  changing  the  temperature  of 
the  others.  There  are  twenty  tubes  for  opsonic  pipettes  and  an  extra  tube.  The  tubes 
may  be  easily  removed  when  desired  by  means  of  a  key  which  accompanies  the  incubator. 
On  top  there  are  eight  tubes,  22  mm.  in  diameter,  for  test-tubes.  Each  is  provided  with  a 
nickel-plated  cap.  The  incubator  is  supplied  with  thermometer,  thermo-regulator,  and 
a  two-flame  burner,  with  wire  guard. 


opsonins  which  induce  phagocytosis.  It  is  furthermore  possible  to  increase 
the  activity  of  these  agents  artificially.  All  three  agents  are  specific  in 
nature  as  already  stated.  Ehrlich  has  attempted  to  explain  the  phenomena 
of  immunity  according  to  his  receptor  or  side  chain  theory  (Seitenketten- 
theorie).  This  theory,  which  is  rather  complex  and  highly  technical,  was 
first  used  to  explain  cell  metabolism.  Hinman's  version  of  the  side  chain 
theory  is  very  simple  and  we  give  it  as  follows:  As  applied  to  immunity 
the  basis  of  the  theory  is  the  conception  of  the  duplex  nature  of  antigens. 


I2O 


PHARMACEUTICAL   BACTERIOLOGY. 


An  antigen  is  a  toxin,  of  bacterial  or  other  origin,  which  has  the  power 
when  introduced  into  the  body,  of  inducing  the  formation  of  specific  anti- 
bodies. Not  all  toxins  or  poisons  have  this  power.  For  example,  strychnin 
and  the  toxin  of  tetanus  iproduce  similar  physiologic  effects,  but  only  the 
latter  is  capable  of  producing  an  antibody.  Ehrlich  explains  this  difference 
by  assuming  that  strychnin  and  most  other  vegetable  poisons  enter  into  a 
loose  combination  with  the  cell  plasm,  analogous  to  an  aniline  dye  which 
can  be  readily  dissolved  out  again;  whereas  the  toxin  is  firmly  bound 
to  the  cell,  representing  in  a  measure  a  toxic  food-stuff  in  chemical 


FIG.  54. — Illustrating  cell  receptors  of  the  first  order.  A  cell  receptor  (a)  uniting 
with  the  haptophore  (c)  of  the  toxin  molecule  or  antigen.  The  toxin  molecule  or  antigen 
consists  of  the  haptophore  and  the  toxophore.  The  toxophore  produces  the  toxic  effects 
upon  the  cell,  e  is  the  haptophore  of  the  cell  receptor  which  has  the  power  of  combin- 
mg  with  the  toxin  molecule  thus  neutralizing  its  possible  toxic  effects.  Free-cell  re- 
ceptors constitute  the  antibodies,  and  are  ever  ready  to  combine  with  antigens  or  toxins, 
should  any  be  present.  Cell  receptors  and  antigen  bodies  are  specific  in  action.  The 
haptophore  of  the  diphtheria  cell  receptor  does  not  fit  the  haptophore  of  tetanus,  for  ex- 
ample. Each  antigen  or  toxin  reacts  with  the  antibodies  fitted  to  it.  (Journal  of  the 
American  Medical  Association,  1905,  p.  955.) 


union  with  and  assimilated  by  the  cell.  The  atomic  combination  of 
the  toxin  antigen,  which  represents  this  chemical  union  is  designated 
the  haptophore  group,  while  the  atomic  combination  of  the  cell-plasm 
with  which  the  haptophore  group  unites  is  called  the  cell  receptor 
group.  The  haptophore  group  is  distinct  from  the  atomic  group  which 
produces  the  toxic  or  pathologic  effects,  designated  as  the  toxophore 
group.  These  two  groups  of  the  antigen  (toxin),  namely,  the  hapto- 
phore group  and  the  toxophore  group,  act  independently  of  each 
other  and  possess  different  properties.  The  toxophore  group  is  easily 


IMMUNITY  AND   IMMUNIZING  AGENTS. 


121 


destroyed  by  heat  (60°  to  65°  C.)  while  the  haptophore  group  is  not  de- 
stroyed, retaining  the  power  of  combining  with  the  receptor  group  of  the 
living  cell.  The  toxophore  group  is  not  necessarily  simple.  LLmay  com- 
prise two  or  more  different  groups.  Snake  poison  contains  two  toxophore 
groups,  one  agglutinating  red  blood  cells,  the  other  causing  its  general  tox- 
icity.  Diphtheria  toxin  also  has  two  toxophore  groups,  the  one  causing 
the  acute  symptoms  and  the  other,  the  toxones  with  a  long  incubation, 
causing  the  later  paralyses  and  cachexias. 

The  nature  of  immunity  to  these  antigens  is  conceived  as  follows:     The 
haptophore  group  is  bound  to  the  cell  receptor  because  of  a  specific  affinity. 


r-f 


FIG.  55. — Illustrating  receptors  of  the  second  order,  Fig.  54,  illustrating  receptors  of 
the  first  order,  c,  d,  The  cell  receptor  with  a  Zymophore  group  (d)  and  a  haptophore 
group  (e)  capable  of  combining  with  disintegrated  bacterial  substances  (/).  The  Zymo- 
phore group  produces  a  ferment  which  acts  upon  (disintegrates)  the  bacterial  cell  or  blood- 
corpuscle,  as  the  case  may  be,  seized  upon  by  the  haptophore  group.  (Journal  of  the 
American  Medical  Association,  1905,  p.  1113.) 

As  a  result  this  particular  side  chain  or  receptor  is  lost  to  the  living  cell  and, 
following  Weigert's  law  of  supercompensation  in  regeneration,  the  cell 
replaces  this  loss  by  producing  many  more  receptor  groups  than  were  pre- 
viously present.  As  in  the  callus  following  a  fracture  there  is  an  over- 
production. In  this  way  such  a  large  number  of  receptors  of  one  type  are 
produced  that  they  become  excessive  and  the  cell  thrusts  them  off  into  the 
blood  and  into  the  fluids  of  the  body.  Here  they  constitute  the  specific 
antibodies  and,  because  of  their  specific  affinity,  unite  with  the  haptophore 
group  of  toxins  and  prevent  their  reaching  the  cell  which  they  thus  protect. 

Therefore,  in  antitoxic  immunity  there  are  three  stages:  First,  the 
chemical  union  of  the  haptophore  group  of  antigen  to  the  receptor  group  of 


122  PHARMACEUTICAL    BACTERIOLOGY. 

the  protoplasm  molecule;  second,  the  overproduction  and  liberation  of  these 
receptors  following  this  binding;  and  third,  the  union  of  these  free  receptors 
or  antibodies  with  free  toxin  haptophore  groups  before  these  can  reach  the 
cell  to  injure  them  by  the  action  of  their  toxophore  groups.  The  antigens 
that  are  known  with  their  respective  antibodies  as  given  by  Hektoen  are: 

Antigens.  Products  of  Immunization. 

Toxins Antitoxins    . 

Ferments Antiferments 

Precipitinogens Precipitins 

Agglutinogens Agglutinins 

Opsonogens Opsonins 

Lysogens Amboceptors  or  lysins 

Antitoxins Antianti  toxins 

Agglutinins Antiagglutinins 

Complements Anticomplements 

Opsonins Antiopsonins 

Amboceptors .  Antiamboceptors 

Precipitins Antiprecipitins 

These  antibodies  all  result  from  the  overproduction  of  simple  receptors, 
but  the  protoplasm  of  cells  may  form  still  other  cell  receptors  which  are  much 
more  complicated  and  subserve  the  absorption  of  more  complicated  and 
complex  albuminous  molecules  than  those  of  toxins. 

Bacterial  clumping  or  agglutinating  phenomena  are  extremely  interesting 
as  well  as  valuable  in  the  diagnosis  of  disease.  Upon  this  behavior  of 
bacteria  depends  the  Widal  typhoid  fever  test.  If  the  serum  of  an  animal 
inoculated  with  the  typhoid  bacillus  (antiserum)  is  added  to  a  liquid  culture 
or  suspension  of  typhoid  bacilli,  the  latter  cease  to  move  and  after  a  time 
become  aggregated  into  irregular  clumps  or  masses.  The  same  phenomenon 
is  observed  if  instead  of  blood  of  a  typhoid  injected  animal,  the  blood  of  a 
typhoid  fever  patient  is  employed.  The  reaction  is  quite  specific,  though 
not  absolutely  so.  That  is,  similar  agglutinating  phenomena  are  produced 
by  related  bacilli,  as  the  typhoid  bacillus,  the  para-typhoid  bacillus  and  the 
colon  bacillus.  Many  other  bacteria,  beside  the  colon-typhoid  group,  are 
agglutinated  by  their  respective  antisera.  In  addition  to  diagnosing  disease 
as  in  typhoid  fever  (the  Widal  test  gives  results  even  before  there  are  marked 
disease  symptoms),  the  agglutinating  phenomena  are  useful  in  the  identifica- 
tion of  bacteria.  The  technic  while  not  difficult,  calls  for  many  pre- 
cautionary measures  and  requires  considerable  time  and  care  to  avoid 
erroneous  conclusions. 

In  1897  Kraus  found  that  when  the  germ-free  filtrates  from  broth  cultures 
of  bacteria  were  mixed  with  their  respective  antisera  (serum  from  animals 
inoculated  with  the  specific  bacteria)  the  formation  of  a  white  precipitate 
occurred.  The  substance  in  the  immunized  serum  which  causes  the  forma- 


IMMUNITY  AND   IMMUNIZING  AGENTS. 


I23 


tion  of  the  precipitate  has  been  termed  precipitin.  Similar  reactions  are 
observed  with  milk  and  egg  albumen,  when  used  with  their  specific  immune 
sera.  These  reactions  have  been  utilized  to  secure  evidence  in  crimina 
cases.  The  serum  of  an  animal  which  has  been  injected  with  human  blood 
(humanized  immune  serum)  produces  a  precipitate  when  mixed  with 
human  blood,  even  in  high  dilutions.  Like  agglutination,  the  reaction  is, 
however,  not  wholly  specific.  For  example,  humanized  animal  serum  will 
also  produce  a  precipitate  with  the  blood  of  higher  apes.  Dog  immunized 
animal  serum  will  produce  a  precipitate  with  wolf's  blood,  etc. 

The  chief  immunizing  agents  are  the  bacterolysins,  the  antitoxins  and 
the  leucocytes  (phagocytes)  aided  by  the  opsonins.     The  significance  of 


FIG.  56. — Illustrating  receptors  of  the  third  order,  or  so-called  amboceptors.  This 
serves  to  explain  the  action  of  lysins  (bacteriolysin,  hemolysin,  cell  lysins,  milk  lysins,  etc.). 
The  cell  receptor  (amboceptor)  has  two  haptophore  groups,  one  (e)  capable  of  uniting 
with  a  disintegrated  substance  as  bacterial  cell,  blood-corpuscle,  etc.,  (/)  and  the  other 
(g)  having  the  power  to  combine  with  a  complement  (&).  h  is  the  haptophore  group  of 
the  complement  (lysin)  and  z  the  zymotoxic  group.  Amboceptors,  lysin  receptors  and 
receptors  of  the  third  order  mean  the  same  thing.  (Journal  of  the  American  Medical 
Association,  1905,  p.  1369.) 

agglutinins  and  precipitins  in  the  prevention  of  bacterial  disease  is  not  clear. 
Recent  observations  on  drug  action  tend  to  prove  that  some  of  these  rem- 
edial agents  apparently  possess  antitoxic  and  other  immunizing  properties. 
It  is  for  example  fairly  well  proven  that  phosphorus  and  Echinacea  angusti- 
folia  have  the  power  of  increasing  the  opsonic  index  in  certain  bacterial  in- 
vasions. Sulphide  of  carbon  and  silica  appear  to  check  and  cure  suppurative 
processes,  perhaps  due  to  similar  activity.  Nuclein  which  is  usually  derived 
from  yeast,  is  reported  to  be  decidedly  bactericidal  and  to  increase  phago- 
cytosis to  a  marked  degree.  According  to  Lloyd,  Lobelia,  when  admin- 
istered hypodermically,  counteracts  the  toxin  of  the  diphtheria  bacillus, 


124 


PHARMACEUTICAL  BACTERIOLOGY. 


being  similar  in  its  effects  to  the  antidiphtheric  serum  (antitoxin  of  diphthe- 
ria). Belladonna  is  reported  to  be  prophylactic  as  well  as  curative  in  scarlet 
fever.  It  is  highly  probable  that  as  our  knowledge  of  the  therapeutic  action 
of  drugs  develops,  there  will  be  a  complete  revolution  in  their  use  as  remedial 
agents. 

In  conclusion,  we  give  a  summarizing  table  of  the  several  immunizing 
agents  above  referred  to  and  which  will  be  more  fully  explained  in  the 
chapter  following. 


Immunizing 
Agents 


Active 


Natural 


Artificial 


f    Bacteriolysins. 

Inherited  or  normal    <j    Opsonins. 
[   Phagocytes. 

Augmented — Immunizing  infectious  diseases. 

f  Toxins. 
Modified  toxins       „  . 

<  Small-pox  vaccination, 
and  toxins.  T.,.  ... 

I  Rbies  vaccination. 


Bacterial 


Bacterins  or  vaccines. 
Bactericidal  sera. 


Passive 


Antitoxins — Diphtheric,  tetanic,  etc. 
Drugs — Nuclein,  lobelia,  phosphorus,  etc. 


CHAPTER  VIII. 
THE  MANUFACTURE  AND  USE  OF  SERA  AND  VACCINES. 

The  most  wonderful  recent  discoveries  in  the  science  of  bacteriology 
pertain  to  the  relationship  of  pathogenic  germs  and  the  serum  of  the  blood 
of  susceptible  animals.  As  already  stated  blood  serum  has  bactericidal 
properties  (see  lysins),  but  it  is  often  not  sufficiently  active  to  destroy  certain 
invading  germs  (pathogenic)  and  the  disease  manifestations,  due  to  the 
toxins  liberated  by  the  germs,  gradually  develop.  The  bacterial  toxins  are 
of  two  kinds,  those  which  escape  from  the  bacterial  cells  and  are  soluble  in 
the  surrounding  media,  entering  the  system  by  absorption;  and  those  which 
remain  within  the  germ  cell  and  are  set  free  only  on  the  breaking  up  of  the 
bacterial  cells.  The  former  are  the  toxins  proper  or  exotoxins,  the  latter 
are  called  endotoxins.  As  already  explained  the  toxins  cause  the  develop- 
ment within  the  serum  of  the  blood  of  certain  substances  (antibodies), 
which  neutralize  or  overcome  the  effects  of  the  toxins  and  which  are  called 
antitoxins.  Investigators  hoped  that  experiments  would  prove  that  every 
pathogenic  germ  would  cause  the  development  of  a  corresponding  antitoxin 
which  might  be  used  in  the  treatment  of  the  disease.  This  hope  has  not 
been  realized.  Of  the  numerous  experimentations  with  antitoxins  only  one 
has  thus  far  proven  entirely  satisfactory,  namely,  the  antitoxin  of  diphtheria. 
Several  others  have  proven  more  or  less  useful,  as  will  be  explained  later, 
but  they  are  far  from  satisfactory. 

The  antitoxins  act  by  neutralizing  the  bacterial  toxins  of  the  disease,  and 
not  by  acting  upon  and  killing  the  germs  themselves.  In  this  regard  the 
antitoxins  or  antitoxic  sera  differ  from  the  antibacterial  or  bactericidal  sera, 
which  act  by  preventing  the  development  of  the  bacteria.  This  distinction 
and  difference  is  not  generally  understood.  The  bactericidal  sera  have, 
however,  thus  far  proven  quite  unsatisfactory  in  the  treatment  of  disease. 
They  are  not  standardized  by  units  as  are  the  antitoxins.  The  dose  is  by 
volume,  from  10  to  50  c.c.,  and  even  more,  usually  given  hypodermically. 
The  sera  are  produced  by  injecting  increasing  amounts  of  germs  (artificially 
cultured)  into  the  animal,  as  the  horse.  As  a  rule  the  first  injections  con- 
sist of  dead  germs;  finally,  living  germs  of  different  virulency  may  be  used. 
By  this  means  a  tolerance  is  established.  The  serum  obtained  from  animals 
thus  immunized  is  used  in  the  treatment  of  disease,  its  action  depending 
upon  its  bactericidal  properties.  There  is  a  group  of  sera  known  as  com- 

125 


126  PHARMACEUTICAL   BACTERIOLOGY. 

posite,  which  give  evidence  of  being  a  decided  improvement  over  the  simple 
sera.  They  are  called  composite  because  they  have  the  peculiar  qualities 
of  two  distinct  forms  of  immunity — for  example,  diphtheria-immune  horses 
may  be  used  in  the  subsequent  bacterial  inoculation,  which  gives  the  result- 
ing immune-serum  a  double  content  of  a  corresponding  antibacterial  body 
and  of  diphtheric  antitoxin.  This  subject  is  as  yet  entirely  in  the  experi- 
mental stage.  It  is  also  known  that  one  kind  or  type  of  immunity  has  some 
influence  not  only  upon  other  immunities,  but  also  upon  other  diseases. 
The  antitoxin  of  diphtheria,  for  example,  appears  to  act  as  a  cure  or  pro- 
phylactic against  pathological  conditions  other  than  diphtheria. 

We  now  come  to  a  third  class  of  substances  used  in  the  treatment  of 
disease,  namely,  the  bacterial  vaccines,  also  designated  bacterins  and  opsono- 
gens  (Ohlmacher).  The  term  vaccine  (from  Vacca,  a  cow)  is  appropriately 
applicable  to  the  small-pox  remedy,  but  is  entirely  inapplicable  to  these 
newer  agents.  Either  bacterin  or  opsonogen  is  a  suitable  name. 

Bacterins  are  simply  suspensions  of  dead  pathogenic  germs  which  are 
used  in  the  treatment  of  disease.  They  produce  their  beneficent  effects  by 
acting  upon  the  bacteria  so  that  they  may  be  taken  up  and  digested  by  the 
white  blood-corpuscles  (phagocytes),  as  has  already  been  explained.  A 
homologous  or  autogenous  bacterin  is  prepared  from  germs  taken  direct 
from  the  patient  and  is  used  in  treating  the  same  patient.  A  heterologous 
bacterin  is  one  which  is  derived  from  a  source  other  than  the  patient  under 
treatment.  A  mixed  bacterin  is  one  in  which  the  germs  (of  the  same  species) 
used  are  derived  from  several  sources.  The  manufactured  bacterins  (heter- 
ologous) ready  for  use  by  the  physician  are  called  stock  vaccines  or  stock 
bacterins. 

The  following  is  a  tabulation  of  antitoxins,  toxins,  antibacterial  sera  and 
bacterins  found  upon  the  market  and  used  by  physicians  and  veterinarians. 

i.  For  Human  Use. 

A.  Antitoxic  Sera  or  Antitoxins. 
Antidiphtheric  serum. 

Liquid  or  usual  form. 

Concentrated  form. 

Dry  form  (official  in  some  pharmacopoeias). 
Antitetanic  serum. 

Liquid  or  usual  form. 

Dry  form. 

B.  Antibacterial  Sera  or  Bactericidal  Sera. 
Antistreptoccocic  serurrL 
Antipneumococcic  serum. 
Antimeningitic  serum. 


THE   MANUFACTURE  AND    USE    OF    SERA  AND   VACCINES.  127 

Antityphoid  serum. 

Antidysenteric  serum. 

Antigonorrheal  serum. 

Antiplague  serum  (Yersin's  serum). 

Antianthrax  serum. 

Scarlet  fever  serum  (Marpmann's  serum). 

Antituberculous  serum  (antituberculins). 

C.  Bacterins  or  Opsonogens.     (Vaccines). 

(Homologous  or  autogenous,  heterogenous  and  mixed.) 
Staphylococcus. 

S.  pyogenes  albeus    ) 

S.  pyogenes  aureus.  \  used  singly  or  mixed. 

S.  pyogenes  citreus.  J 
Streptococcus. 
Gonococcus. 
Typhoid. 

Typhoid  (Shafer's  mixed  bacterin). 
Colon  bacillus. 
Neoformans  bacillus. 
Pyocyaneous  bacillus. 

Bubonic  plague  bacillus  (Haffkine's  plague  vaccine). 
Tuberculins. 

Tuberculin,  old  (T.  O.). 

Tuberculin  residuum  (T.  R.). 

Tuberculin  precipitate  (T.  P.). 

Bacillus  emulsion  (B.  E.). 

Bacillus  filtrate  (B.  F.). 

D.  Toxins  (modified). 

Small-pox  vaccine. 

On  ivory  points. 

In  glycerinated  tubes. 

Dry  form. 

Hydrophobia  vaccine. 
Erysipelas  and  Prodigiosus  toxin.     (Cancer  and  other  malignant 

growths) . 

Antivenine.     (Snake  toxin.) 
Cancer  vaccin  (Oilman's  vaccine). 

2.  For  Veterinary  Use. 

A.  Antitoxic  Sera  or  Antitoxins. 
Antitetanic  serum. 
Influenza  serum.     (Intravenous  use.) 


128  PHARMACEUTICAL  BACTERIOLOGY. 

B.  Antibacterial  Sera  or  Bactericidal  Sera. 

Antistreptococcic  serum. 
Canine  distemper  serum. 
White  scour  serum. 

C.  Bacterins. 

Anthrax. 

Mallein. 

Tuberculin. 

Blackleg. 

Blacklegine. 

Blacklegules  (pill  form). 

Blacklegoids  (pill  form). 

Hog  cholera. 

Fowl  cholera. 

White  scour. 

Texas  fever. 

The  above  substances  resemble  each  other  in  that  they  are  organic  and 
of  complex  chemical  composition.  They  gradually  deteriorate  and  finally 
become  worthless,  some  sooner  than  others.  Even  the  comparatively  per- 
manent kinds  will  not  retain  their  full  properties  more  than  a  few  months, 
though  they  may  still  be  sufficiently  active  therapeutically  after  eighteen 
months  or  even  longer.  They  should  be  kept  in  a  cool  dry  place,  away  from 
light.  Turbidity  in  those  preparations,  which  are  clear  when  freshly  pre- 
pared, indicates  that  decomposition  changes  have  set  in  and  that  they  are 
unfit  for  use.  Many  of  the  bacterins  are  normally  turbid  and  nearly  all  of 
them  have  some  slight  color  and  odor. 

Thus  far  only  a  few  of  the  substances  above  tabulated  have  proven 
entirely  satisfactory  in  the  treatment  of  the  particular  disease  or  diseases 
for  which  they  were  intended.  This  is  but  to  be  expected  since  their  use  is 
very  largely  based  upon  theory.  Theory  and  practice  have  ever  failed  to 
develop  along  exactly  parallel  lines.  Science  is  however  fortunate  in  being 
able  to  assert  that  in  the  antidiphtheric  serum  we  have  practically  a  specific 
for  the  cure  of  diphtheria,  provided  it  is  used  in  time  and  given  in  sufficiently 
large  and  sufficiently  frequent  doses.  The  anti tetanic  serum  has  given 
excellent  results  particularly  as  a  preventive,  as  has  also  the  antistreptococcic 
serum.  Of  the  bacterins  the  Staphylococcus  has  given  excellent  results  in 
the  cure  of  actual  pathologic  conditions.  Some  of  the  others  have  proven 
less  satisfactory  and  in  many  cases  their  great  usefulness  lies  in  their  prevent- 
ive rather  than  curative  powers.  The  tuberculins,  in  particular,  give  promise 
of  great  usefulness  in  the  eradication  of  the  dread  white  plague. 

We  will  explain  very  briefly  the  manufacture  of  •&  few  of  these  substances 
only,  as  the  methods  are  quite  closely  similar  for  like  agents.  The  following 


THE    MANUFACTURE    AND    USE    OF    SERA   AND    VACCINES.          I2Q 

is  a  brief  outline  of  the  manufacture  of  the  marvelous  remedy  for  the  treat- 
ment of  the  dread  disease  of  childhood,  namely  diptheria. 

3.  Antidiphtheric  Serum. 

A.  Selecting  and  Testing  the  Horse. — Ordinary,  normal,  non-pedigree 
horses  are  preferred,  purchased  under  a  guarantee  of  soundness.     Even 
though  purchased  under  such  a  guarantee  the  animal  is  kept  under  observa- 
tion for  a  few  weeks  and  tested  for  glanders  by  the  mallein  test.     No  animal 
is  retained  until  it  is  proven  that  there  is  no  latent  or  active  disease  present. 
The  animal  is  well  housed  and  well  cared  for  during  the  entire  time,  under 
conditions  as  sanitary  as  it  is  possible  to  make  them.     All  laboratories  are 
also  regularly  visited  by  a  U.  S.  Government  inspector,  who  reports  his  find- 
ings to  Washington. 

B.  Preparing  the  Toxin  of  Diphtheria. — Pure  cultures  of  a  breed  or 
strand  of  the  diptheria  bacillus,  possessed  of  a  high  potency,  virulency  or 
toxicity,  are  made  in  liter  flasks  containing  beef  bouillon.     The  original  ba- 
cilli thus  used  are  taken  from  some  patient  suffering  with  diptheria,  and  by 
means  of  isolation  methods  all  foreign  microbes  are  rejected  or  excluded. 
After  the  culture  is  several  days  old  or  when  a  maximum  amount  of  the  toxin 
has  been  formed  and  deposited  in  the  bouillon,  the  bacilli  are  killed  by  adding 
0.25  per  cent,  of  trikresol.     The  bouillon  with  the  dead  bacilli  is  filtered. 
The  clear  filtered  substance  constitutes  the  toxin  which  is  injected  into  the 
horse  for  the  purpose  of  developing  (in  the  horse)  the  antitoxin  of  diptheria. 
The  virulency  or  potency  of  the  toxin  varies  and  is  tested  on  guinea-pigs  and 
compared  with  the  U.  S.  Government  standard.     The  desirable  breed,  race 
or  strain  of  germs  is  perpetuated  in  the  laboratory  by  daily  transfers  to  new 
culture  tubes.     In  this  manner  the  bacilli  are  maintained  for  a  long  time, 
several  years  or  longer.     However,  even  with  the  greatest  care  the  race  finally 
deteriorates,  weakens  or  undergoes  a  change  in  potency  and  it  becomes 
necessary  to  secure  a  new  stock  culture. 

C.  Developing  the  Antitoxin  of  Diptheria  in  the  Horse. — Twice  weekly 
the  horse  is  given  (by  hypodermic  injection  into  the  flank  region)  gradually 
increasing  doses  of  the  toxin  of  diphtheria.     The  rule  is  to  give  enough  to 
produce  a  marked  reaction.     For  a  day  or  two  the  horse  is  sick  with  diph- 
theria, then  recovers  as  the  increased  antitoxin  in  the  blood  (serum)  of  the 
animal  neutralizes  the  toxin.     This  is  continued  for  from  four  to  six  weeks 
when  a  maximum  amount  of  antitoxin  has  presumably  developed.     The 
last  dose  of  toxin  is  several  hundred  times  greater  than  the  first. 

D.  Bleeding  the  Horse. — A  sterilized  canula  or  trochar  is  inserted  into 
the  jugular  vein,  after  the  neck  has  been  thoroughly  washed  with  soap  and 
water,  shaved  and  rinsed  with  a  5  per  cent,  solution  of  carbolic  acid.     The 

9 


130  PHARMACEUTICAL    BACTERIOLOGY. 

blood  is  drawn  off  into  sterilized  liter  tubes,  which  are  plugged  with  cotton. 
From  nine  to  twelve  liter  of  blood  are  taken  from  the  horse  at  one  time  and 
the  bleeding  is  repeated  four  or  five  times  at  intervals  of  about  six  months. 
The  punctured  wound  is  closed  by  keeping  an  artery  forceps  in  position  for 
a  short  time. 

E.  Securing  the  Serum. — The  blood  tubes  are  set  aside  until  the  clot  has 
formed  and  settled  to  the  bottom.  The  clear  serum  is  siphoned  off  into  a 
large  flask,  0.25  per  cent,  of  trikresol  is  added  as  a  preservative  and  to  kill 


FlG.  57. — Bleeding  the  horse  after  a  maximum  amount  of  the  antitoxin  of  diphtheria  has 
been  developed  in  the  blood.     The  animals  pay  but  little  attention  to  the  operation. 

any  germs  that  might  be  accidentally  present,  and  then  filtered  through 
several  thicknesses  of  filter  paper,  under  pressure  (suction).  The  perfectly 
clear,  sterile  and  germ-free  serum  constitutes  the  antitoxin  of  diphtheria  and 
is  ready  for  use  as  soon  as  it  is  standardized  and  put  into  suitable  containers. 
F.  Standardizing  the  Antitoxin  of  Diphtheria. — Since  the  antitoxic  valence 
of  horse  serum  as  above  described  varies  somewhat,  it  is  necessary  to  deter- 
mine the  quantitative  value  in  order  that  physicians  may  know  what  amounts 
to  administer  in  the  treatment  of  diphtheria.  The  standard  unit  of  strength 


THE   MANUFACTURE  AND    USE    OF    SERA  AND    VACCINES.  131 

now  adopted  by  all  civilized  countries  is  the  so-called  Ehrlich  unit,  which 
is  the  amount  of  serum  (antitoxin  of  the  horse)  which  will  just  neutralize 
one  hundred  times  a  fatal  dose  of  toxin  when  administered  to  a  guinea-pig, 
weighing  approximately  250  grams  or  one-half  pound.  The  U.  S.  standard 
is  prepared  in  the  biological  laboratories  of  the  U.  S.  Marine  Hospital  Service 


FIG.  58. — Sterilized  liter  tubes  into  which  the  blood  drawn  from  the  horse  is  placed. 
The  top,  covered  with  sterilized  cloth,  is  connected  with  the  canula  in  the  jugular  vein 
of  the  animal. 

at  Washington,  and  every  manufacturer  of  diphtheric  antitoxin  in  the  United 
States  is  supplied  with  standard  units  from  this  laboratory.  The  method 
of  procedure  is  approximately  as  follows:  Eight  containers  (test-tubes)  are 
set  out  in  a  row  and  numbered  or  marked  serially.  Into  each  tube  is  poured 
just  one  hundred  fatal  doses  of  toxin  (fatal  to  a  250  gram  guinea-pig,  deter-- 


132 


PHARMACEUTICAL   BACTERIOLOGY. 


mined  experimentally),  and  a  graded  amount  of  the  serum  to  be  standard- 
ized/so that  the  first  tube  has,  in  all  probability,  not  enough  antitoxin  to 
neutralize  the  one  hundred  fatal  doses  of  the  toxin,  and  the  eighth  tube  has, 
in  all  probability,  a  great  excess  of  antitoxin.  The  contents  of  one  tube  is 
injected  into  a  guinea-pig,  thus  requiring  eight  pigs.  The  animals  are 
marked  and  kept  under  close  observation.  The  first,  second  and  perhaps 
third  die,  showing  that  not  enough  serum  was  added  to  neutralize  the  toxin. 
The  fourth  pig  just  recovers,  showing  that  the  amount  of  serum  added  to  the 
fourth  tube  was  sufficient  to  neutralize  one  hundred  fatal  doses  of  the  toxin. 


FIG.  59. — Guinea-pigs  in  wire  cages.  These  lively  little  animals  are  used  in  testing  the 
virulense  of  the  diphtheria  toxin  which  is  injected  into  the  horse  and  also  for  the  purpose 
of  standardizing  the  antitoxin.  The  reasons  why  these  animals  are  preferred  are  wholly 
biological  and  physiological.  They  propagate  rapidily,  are  easily  kept,  easily  handled, 
and  respond  (biologically)  to  the  tests  applied. 


This  amount  of  serum  (antitoxic)  represents  one  unit.  From  this  amount 
or  unit  the  quantities  to  be  put  into  the  containers  are  determined.  500, 
1000,  2500  and  5000  unit  quantities  are  put  up,  for  the  convenience  of 
physicians.  500  units  constitute  an  immunizing  dose,  given  to  those  who 
do  not  have  diphtheria,  but  who  have  been  exposed  to  the  disease.  The 
larger  doses  are  curative.  The  rule  is  to  give  large  doses,  repeated  as  often 
as  may  be  necessary. 


THE   MANUFACTURE  AND   USE    OF   SERA  AND   VACCINES. 

4.  Concentrated  Diphtheric  Antitoxin, 


133 


While  the  chemical  nature  of  antitoxin  is  not  known,  it  has  been  deter- 
mined that  it  is  united,  in  some  way,  with  the  globulins  of  the  blood.  The 
attempts  to  isolate  antitoxin  have  resulted  in  the  manufacture  of  a  refined  or 
concentrated  antidiphtheric  serum  which  is  used  quite  extensively  though 
it  does  not  meet  with  the  unqualified  favor  accorded  the  antidiphtheric 
serum.  The  process  of  manufacture  is  as  follows: 


FIG.  60. — Container  with  diphtheria  antitoxin,  supplied  with  hypodermic  needle,  piston, 
all  ready  for  immediate  use  by  the  physician.  The  plunger  is  simply  a  homeopathic  vial 
with  rubber  stopper.  (Cutter  Laboratory.} 

A.  The   antidiphtheric   serum  is   saturated   with   ammonium   sulphate 
which  precipitates  the  globulins  (containing  the  antitoxin)  in  the  form  of  a 
white  mass.     It  is  then  filtered  and  the  filtrate  rejected. 

B.  The  precipitate  left  on  the  filter  is  redissolved  in  water  and  this  solu- 
tion is  again  treated  with  ammonium  sulphate  as  in  (a).     The  object  in 
redissolving  in  water  is  to  wash  the  globulins. 


134  PHARMACEUTICAL    BACTERIOLOGY. 

C.  The  second  precipitation  product  is  treated  with  a  saturated  salt 
solution  which   dissolves  the  antitoxin  globulins.     The  solution  is  then 
filtered. 

D.  To  the  filtered  solution  2.5  per  cent,  of  acetic  acid  is  added  which 
again  precipitates  the  globulins  on  the  filter  paper  where  it  is  partially  dried 
by  means  of  filter  paper  and  towels  pressed  upon  the  mass. 

E.  The  partially  dried  material  is  placed  in  a  dialyzing  bag  and  suspended 
in  a  water  current,  for  several  days.     This  removed  the  salts  by  osmotic 
action  and  at  the  same  time  the  globulins  enter  into  solution  within  the  bag. 

F.  A  preservative  is  added  to  the  liquid  and  which  is  then  passed  through 
a  Berkefeld  filter.     Some  physiologic  salt  solution  is  also  added.     This  is 
the  final  product. 

G.  .After  being  tested  bacteriologically  to  make  sure  that  it  is  not  con- 
taminated, it  is  standardized  as  described  under  diphtheric  serum. 

The  above  process  removes  the  following  non-active  substances:  serum 
albumins,  lecithin,  cholesterin,  traces  of  bile  salts  and  acids,  blood  salts 
and  the  non-antitoxic  globulins.  The  dosage  of  the  concentrated  antitoxin 
is  less  than  that  of  the  non-concentrated  serum  and  it  keeps  longer.  For 
the  manufacture  of  the  concentrated  diphtheria  antitoxin  the  returned 
serum  is  generally  employed,  that  is  serum  which  has  exceeded  the  time 
limit  of  use. 

5.  Antitetanic  Serum. 

This  is  prepared  similarly  to  antidiphtheric  serum.  The  tetanus  bacilli 
are  grown  in  bouillon,  in  the  absence  of  oxygen,  since  tetanus  germs  are 
anaerobic.  The  growth  is  then  killed,  filtered  out  and  the  clear  toxic,  germ- 
free  bouillon  filtrate  is  utilized  in  the  immunization  of  the  horse.  Small  doses, 
usually  mixed  with  some  antitetanic  serum,  are  administered  at  first  and 
gradually  increasing  the  amount  as  the  horse  can  stand  it  until  large  quantities 
are  given,  even  as  much  as  700  or  800  c.c.  After  some  months  the  horse  is 
bled  in  the  same  manner  as  for  antidiphtheric  serum,  the  serum  is  separated 
and  bacteriologically  tested  in  the  same  way. 

The  unit  of  tetanus  antitoxin  is  that  quantity  of  antitetanic  serum  which 
is  necessary  to  completely  neutralize  1000  fatal  doses  of  tetanus  toxin  for  a 
25o-gram  guinea-pig. 

Antitetanic  serum  has  not  been  a  marked  success  as  a  curative  agent.  Its 
greatest  usefulness  appears  to  be  as  a  prophylactic,  for  which  purpose  it 
should  be  given  early,  as  soon  as  the  injury  (cut,  gunshot  wound,  abrasion) 
has  occurred. 

The  following  are  the  more  important  antibacterial  sera.  A  fuller 
description  of  the  processes  of  manufacture  is  omitted  as  that  is  a  matter  of 


THE   MANUFACTURE  AND    USE    OF   SERA  AND   VACCINES.  135 

no  special  importance  to  the  pharmacist.     Furthermore,  manufacturers  do 
not,  as  a  rule,  disclose  full  details  of  manufacture. 

6.     Antipneumococcic  Serum. 

This  serum  is  obtained  from  horses  immunized  against  the  Pneumococcus 
and  is  employed  in  the  treatment  of  pneumonia  and  other  infectious  disease 
in  which  this  germ  is  present.  The  dose  is  about  10  c.c.  repeated  several 
times  a  day,  given  hypodermically.  The  serum  must  be  kept  in  a  cool  dark 
place.  When  a  tube  is  opened  the  contents  should  be  used  within  twenty- 
four  hours,  sealed  temporarily  with  sealing  wax,  paraffin  or  sterile  wadding. 
This  serum  has  not  proven  very  satisfactory,  though  it  is  safe  and  worthy  a 
trial.  (See  pneumonia.) 

7.  Antimeningococcic  Serum. 

Antimeningococcic  serum  is  obtained  from  horses  which  have  been 
immunized  with  cultures  of  Diplococcus  meningitidis  intracellularis,  begin- 
ning with  dead  cultures,  then  using  living  cultures  and  finally  with  autolysate. 
Its  use  is  said  to  have  met  with  considerable  success  in  the  treatment  of 
cerebro-spinal  meningitis,  when  injected  into  the  spinal  canal  in  doses  of  10 
c.c.,  repeated  daily.  The  serum  acts  as  an  antitoxin,  it  increases  phagocytosis 
and  also  acts  as  a  bactericide.  It  should  be  used  early  in  the  course  of  the 
disease. 

8.  Yersin's  Serum  (Antiplague  Serum). 

Yersin's  serum  is  made  by  injecting  horses,  first  with  dead  plague 
bacillus  cultures  (Bacillus  pestis)  and  finally  with  the  living  organisms.  It 
has  been  used  with  varying  success  in  plague  epidemics.  Large  doses  (30  to 
50  c.c.)  should  be  administered  (hypodermically)  early  in  the  course  of  the 
disease.  Its  chief  value  is,  however,  prophylactic.  The  liquid  form  of  the 
serum  may  also  be  used  for  intravenous  injection.  The  dry  serum  is  said 
to  keep  indefinitely  and  must  be  dissolved  before  using. 

9.  Bacterins. 

The  bacterins  are  still,  so  to  speak,  on  trial.  Some  have  given  excellent 
results  while  others  are  wholly  unsatisfactory.  The  preference  appears  to 
be  for  autogenous  bacterins.  The  majority  of  physicians  are,  however, 
compelled  to  use  the  so-called  stock  bacterins,  or  the  manufactured  bac- 
terins ready  for  use,  for  the  reason  that  few  physicians  have  the  time  or 


136  PHARMACEUTICAL   BACTERIOLOGY. 

the  equipment  to  prepare  the  homologous  or  autogenous  bacterins.     The 
method  of  preparing  a  homologous  bacterin  may  be  outlined  as  follows: 

a.  A  tube,  flask  or  plate  with  the  suitable  culture  medium  (agar  or 
gelatin)  is  inoculated  with  the  germs  taken  from  the  patient  and  incubated, 
until  a  maximum  development  has  taken  place,  about  twenty-four  hours. 

b.  The  growth  is  separated  from  the  culture  medium  by  means  of  a 
sterile  physiological   salt  solution  and  a  platinum  wire  loop.     The  salt 
solution  with  the  bacteria  is  transferred  to  a  sterile  test-tube  which  is  then 
sealed  in  a  flame. 

c.  When  the  tube  is  cool,  it  is  shaken  vigorously  so  as  to  emulsify  the 
bacteria  in  the  salt  solution. 

d.  The  tube  is  opened  and  about  one  drop  is  removed  with  which  to 
make  the  blood-corpuscle  count,  to  be  explained  later.     The  tube  is  again 
sealed  in  the  flame. 

e.  The  tube  is  now  placed  in  a  water  bath  (opsonic  incubator  of  special 
construction  for  this  work)  at  a  temperature  of  60°  C.  for  a  sufficient  length 
of  time  to  kill  the  germs;  one  hour  is  usually  adequate.     This  constitutes  the 
bacterin  and  is  ready  for  use  as  soon  as  it  is  standardized.     Usually  some 
preservative  is  added  when  the  tube  is  opened  and  before  the  bacterin  is 
injected  (0.2  per  cent,  lysol,  0.4  per  cent,  trikresol,  etc.). 

f.  From  the  above  it  must  be  evident  that  no  two  preparations  contain 
the  same  number  of  germs  per  c.c.  and  hence  the  physician  cannot  know 
how  many  dead  microbes  are  injected  at  a  dose.     Therefore  the  necessity 
of  standardizing  the  bacterin,  which  is  done  as  follows: 

g.  Mix  one  part  of  freshly  drawn  blood  with  one  part  of  the  bacterin 
(taken  from  the  tube  in  d.),  add  two  or  three  parts  of  physiological  salt 
solution,  and  spread  evenly  on  a  slide.     Examine  under  the  microscope  and 
determine  the  number  of  microbes  per  c.c.  in  terms  of  the  number  of  red 
blood-corpuscles  per  c.c.     This  is  done  by  making  numerous  (10  to  20)  counts 
of  red  blood-corpuscles  and  microbes.     Knowing  that  there  are  5,000,000,000 
red  blood-corpuscles  per  c.c.,  it  is  then  a  simple  matter  to  compute  the 
number  of  microbes  per  c.c.  in   the   bacterin   under   consideration.     The 
count  thus  determined  divided  by  the  number  of  bacteria  desired  for  one 
dose,  indicates  the  number  of  times  the  bacterin  is  to  be  diluted.     This  is 
very  clearly  illustrated  in  a  chart  prepared  by  Hough  ton,  shown  in  Fig.  61. 

The  number  of  bacteria  administered  per  dose  depends  upon  the  thera- 
peutic effects  to  be  produced,  the  kind  of  bacterin  used,  the  nature  of  the  dis- 
ease and  the  condition  of  the  patient.  The  rule  is  to  start  with  small  doses, 
gradually  increasing  them  in  such  a  manner  as  to  secure  a  maximum  of 
positive  opsonic  phases  with  a  minimum  of  negative  opsonic  phases.  In 
round  numbers  the  dosage  ranges  from  5,000,000  to  50,000,000  bacilli,  rep- 
resented by  varying  quantities  of  the  bacterins. 


THE    MANUFACTURE   AND    USE    OF    SERA  AND    VACCINES. 


137 


FIG.  61. — Counting  the  bacteria  in  standardizing  bacterins.  This  chart  shows  the 
count  of  bacteria  and  of  red  blood  cells  in  twenty  successive  fields  of  the  microscope.  The 
number  of  red  cells  counted  (308)  is  to  the  number  of  bacteria  counted  (224)  as  the  number 
of  red  cells  per  cubic  centimeter  in  normal  blood  (5,000,000,000)  is  to  the  number  of 
bacteria  per  c.c.  in  the  suspension  (3,636,000).  This  count  (3,636,000)  divided  by  the 
count  desired  in  the  final  dilution  (400,000,000)  gives  the  number  of  times  (9)  this  sus- 
pension must  be  diluted  to  bring  it  to  the  desired  dilution.  (Parke,  Davis  6r»  Co.). 


138  PHARMACEUTICAL   BACTERIOLOGY. 

10.  Tuberculins. 

The  tuberculins  are  of  special  interest  as  they  give  great  promise  in  the 
successful  treatment  of  tuberculosis.  The  different  kinds  have  their  special 
use.  Their  manufacture  is  briefly  outlined  as  follows: 

A.  Tuberculin  Old  (T.  O.). — This  is  the  original  Koch  tuberculin  or 
Koch  lymph  and  is  a  concentrated  bouillon  culture  of  the  tubercle  bacillus, 
which  has  been  filtered  to  remove  the  germs.     It  is  a  toxin  solution  and 
not  a  bacterin  proper. 

B.  Tuberculin  Residuum  (T.  R.). — This  is  prepared  by  grinding  the 
dried  tubercle  bacilli,  extracting  with  water,  centrifugalizing,  discarding  the 
supernatant  liquid,  regrinding  the  sediment,  which  is  first  allowed  to  dry, 
and  mixing  with  glycerin  and  water.     It  is  thus  a  suspension  of  pulverized 
tubercle  bacilli  in  an  aqueous  solution  of  glycerin.     The  grinding  process 
is  tedious  and  requires  much  time.     The  tuberculin  is  standardized  so  that 
i  c.c.  will  represent  10  mg.  of  the  dry  culture. 

The  supernatant  liquid,  after  centrifugalizing,  is  sometimes  drawn  off, 
instead  of  rejecting,  and  constitutes  the  upper  tuberculin  (T.  O.)  (Obere 
Tuberculin).  These  two  tuberculins  (the  T.  R.  and  the  T.  O.)  differ  in 
therapeutic  value  and  in  physical  properties. 

C.  Bacillus  Emulsion   (B.   E.). — This  consists  of  pulverized   tubercle 
bacilli  suspended  in  50  per  cent,  glycerin  and  is  standardized  to  contain  5  mg. 
of  solid  matter  per  c.c.     It  differs  from  T.  R.  in  that  the  supernatant  liquid 
(T.  O.)  is  not  drawn  off. 

D.  Tuberculin  Precipitate  (T.  R.). — This  is  obtained  from  old  tuberculin 
by  precipitation  with  alcohol,  drying  and  pulverizing  the  precipitate.     It  is 
used  in  making  the  Calmette  eye-test.     (See  tuberculosis.) 

E.  Bouillon  Filtrate     (Tuberculin  Filtrate  B.  F.—Denys  Tuberculin).— 
The  tubercle  bacillus  cultures  are  passed  through  a  Berkefeld  filter  to 
remove  all  germs.     The  filtrate  is  preserved  with  trikresol. 

ii.  Small-pox  Vaccine. 

Small-pox  vaccine  is  not  a  true  toxin  nor  yet  a  true  bacterin.  Its  value 
in  the  eradication  of  small-pox  has  world-wide  recognition.  The  following 
is  the  manner  in  which  small-pox  vaccine  is  prepared. 

A.  Selecting  the  Animal. — A  young  heifer  (five  to  ten  months  old)  is 
selected,  tested  for  tuberculosis  by  means  of  tuberculin.     The  animal  is 
observed  for  a  time  to  make  sure  of  general  condition  of  health ;  is  well  fed 
and  well  cared  for,  under  conditions  as  sanitary  as  it  is  possible  to  keep  them. 

B.  Inoculating  the  Animal. — The  heifer  is  strapped  securely  to  a  frame- 
work, back  down,  the  udder  region  is  cleansed,  shaven  and  cross  marked 
(scarified)  with  a  sharp  scalpel.     The  cuts  are  just  deep  enough  to  cause  the 


THE   MANUFACTURE  AND    USE    OF   SERA  AND   VACCINES. 


139 


escape  of  serum,  not  actual  bleeding.  This  scarified  surface  is  then  inoc- 
ulated with  glycerinated  small-pox  virus  taken  from  a  patient.  When  the 
inoculated  material  has  had  time  to  be  absorbed  the  animal  is  righted  again 
and  cared  for  under  as  aseptic  conditions  as  possible.  In  time  (six  to 
seven  days)  pustules  form  over  the  entire  inoculated  area.  The  virulent 
virus  from  man  conveys  the  disease  to  the  animal,  but  in  its  passage  through 
the  animal  it  becomes  modified,  losing  in  virulency,  yet  capable  of  producing 
immunity  as  the  result  of  a  mild  intoxication  (vaccinia). 

C.  Removing  the  Scab. — The  animal  is  again  fastened  to  the  frame. 


FIG.  62. — Showing  the  heifer  strapped  to  the  frame,  preparatory  to  removing  the 
vaccinia  scab  from  the  area  which  was  scarified  and  inoculated  with  the  small-pox  virus. 
The  scab  patches  show  dark. 

The  inoculated  surface  is  washed  and  dried.  The  thick  scab  which  has 
formed  over  the  inoculated  area  is  then  removed  and  triturated  with  50  per 
cent,  glycerin.  This  constitutes  the  small-pox  vaccine. 

D.  Aging  or  Ripening  the  Vaccine. — The  fresh  or  raw  vaccine  is  not  used 
as  it  contains  various  living  microbes.     It  is  acted  upon  by  the  glycerin 
added,  for  five  or  six  weeks.     The  virus  is  tested  bacteriologically  during 
this  period,  and  as  soon  as  no  more  colonies  appear  it  is  ready  for  use. 

E.  Preparing  for  the  Market. — The  vaccine  is  now  put  into  small  glass 


140  PHARMACEUTICAL   BACTERIOLOGY. 

tubes  and  marketed  as  glycerinated  tube  virus.  The  vaccine  should  be  kept, 
in  a  cool,  dark,  dry  place.  It  deteriorates  gradually  and  the  time  limit  of 
usefulness  is  stamped  on  each  package. 

The  old  time  ivory  tips  are  still  on  the  market  and  are  preferred  by  many 
physicians.  A  dry  bulk  form  of  the  virus  is  also  marketed.  The  manner 
of  the  use  and  the  action  of  the  virus  are  universally  known.  As  now  pre- 
pared the  remedy  is  absolutely  safe.  No  ill  effects  ever  follow  its  use.  Of 
the  millions  of  persons  inoculated  within  recent  years,  there  probably  has 
not  been  a  single  instance  of  bad  effects  which  could  be  traced  primarily 
to  the  vaccine  virus  itself.  A  small-pox  vaccination  is  not  nearly  as  likely 
to  produce  ill  effects  as  the  customary  hand  shake.  In  fact  the  latter  opera- 
tion does  occasionally  spread  an  infection. 

12.  Hydrophobia  or  Rabies  Vaccine. 

Pasteur's  hydrophobia  virus  is  obtained  from  the  spinal  cord  of  rabbits, 
inoculated  with  the  virus  from  a  dog  suffering  with  rabies.  The  inocula- 
tion is  made  into  the  dura  mater  of  the  spinal  cord.  The  rabbit  dies  in 
about  two  weeks.  A  second  rabbit  is  inoculated  from  the  first,  which  dies 
even  sooner,  showing  that  the  toxin  gained  in  virulency  in  its  passage  through 
the  first  animal.  This  is  repeated  until  finally  the  animal  dies  in  six  or  seven 
days  after  inoculation.  Beyond  this  the  virulency  of  the  poison  cannot  be 
increased  and  this  constitutes  the  virus  fixe  (fixed  or  unchanged  virus)  of 
Pasteur. 

The  spinal  cord  of  the  rabbit  dead  of  -virus  fixe  is  dried  in  a  glass  cylinder 
with  potassium  hydrate.  The  cylinder  is  placed  in  a  cool  dry  place  and 
each  day  small  bits  of  the  cord  are  cut  off  and  placed  in  a  vial  of  glycerin. 
At  the  end  of  fourteen  days  the  virus  is  no  longer  capable  of  producing 
hydrophobia  in  rabbits,  but  the  animal  inoculated  with  it  can  withstand 
the  thirteen  days  virus  (which  was  preserved  in  the  glycerin)  and  so  on 
down  the  scale,  until 'finally  the  rabbit  can  withstand  the  virus  fixe  without 
experiencing  serious  effects. 

In  man  it  is  customary  to  begin  the  treatment  for  rabies  (or  suspected 
rabies)  with  the  nine  day  cord  (hypodermic  injections  of  the  cord  emulsions) 
and  to  give  each  succeeding  day  a  virus  one  day  stronger,  until  finally  the 
virus  fixe  is  injected  without  producing  untoward  symptoms.  The  individual 
thus  treated  is  now  able  to  withstand  the  much  weaker  virus  from  a  dog 
or  other  animal  suffering  from  rabies.  As  the  result  of  this  mode  of  treat- 
ment the  mortality  rate  from  rabies  is  now  less  than  i  per  cent.  (Ravenel). 
Those  bitten  by  dogs  (or  wolves,  skunks,  cats)  suffering  from  rabies  or 
suspected  of  suffering  from  rabies,  should  cleanse,  cauterize  and  disinfect 
the  wound  at  once,  and  then  immediately  proceed  to  a  Pasteur  Institute  and 


THE    MANUFACTURE   AND    USE    OF    SERA   AND    VACCINES.          141 

submit  themselves  for  treatment.  The  earlier,  after  infection,  the  treatment 
is  begun  the  more  likely  will  the  results  be  satisfactory.  The  vaccine  is, 
however,  now  so  prepared  as  to  make  home  treatment  possible:  The 
graded  doses  of  the  virus  put  up  in  sterilized  ampuls  are  ready  for  imme- 
diate use  by  the  family  physician. 

13.  Streptococcus  Mixed  Vaccine. 

A  mixed  toxin  of  erysipelas  (Streptococcus  pyogenes  and  Bacillus  pro- 
digiosus}  is  used  in  the  treatment  of  cancer  and  other  malignant  tumors, 
particularly  the  sarcomas.  It  appears  to  have  a  local  as  well  as  systemic 
effect.  It  is  prepared  as  follows:  Virulent  cultures  of  the  Streptococcus 
pyogenes  are  grown  in  the  incubator  for  three  weeks,  then  inoculated  with 
the  Bacillus  prodigiosus  and  allowed  to  grow  ten  days  longer  at  the  room 
temperature.  The  mixed  cultures  are  then  bottled,  sterilized  by  heating 
for  an  hour  at  60°  C.,  and  are  ready  for  use.  Being  unfiltered,  the  prepara- 
tion is  decidedly  turbid  in  appearance.  This  preparation  is  administered 
hypodermically,  the  injection  being  made  in  the  neighborhood  of  the  tumor 
or  into  the  tumor  itself. 

14.  Cancer  Vaccine. 

Recently  Dr.  Gilman  of  Johns  Hopkins  University  has  experimented  with 
a  cancer  vaccine  or  cancer  emulsion  which  it  is  believed  will  cure  cancer. 
It  is  simply  a  preparation  made  from  the  cancerous  tissue  of  the  patient, 
macerated  in  a  physiologic  salt  solution  and  preserved  in  some  antiseptic. 
This  is  suitably  diluted  and  given  in  10  c.c.  doses  injected  directly  into 
the  circulation.  A  reaction  (rise  in  temperature  to  102°  to  104°  F.),  de- 
velops in  three  days,  suppuration  ceases  and  the  tissues  begin  to  heal.  The 
dose  is  repeated  three  times  at  intervals  of  two  weeks  which  is  supposed 
to  be  sufficient  to  effect  a  cure.  Although  not  a  bacterin  as  far  as  can 
be  ascertained,  its  action  is  evidently  similar  to  that  of  an  autogenous 
bacterin.  As  yet  it  is  too  early  in  the  history  of  the  use  of  this  remedy  to 
predict  the  final  results. 


CHAPTER  IX. 
YEASTS  AND  MOULDS. 


The  organisms  commonly  designated  as  yeasts  and  moulds,  though  not 
belonging  to  the  bacteria  (Schizomycetes),  are  of  the  greatest  importance  in 
human  economy  and  play  a  most  active  part  in  life.  Some  of  them  are  most 


FIG.  63. — Development  of  Mucor  mucedo.  a,  6,  c,  d,  stages  in  the  formation  of  the 
zygospore;  d,  mature  zygospore;  e,f,  endospore  formation;  g,  endospores;  h,  germinating 
spore,  this  develops  and  finally  gives  rise  to  new  zygospores;  i,  mucor  slightly  magnified. 
This  mould  is  found  on  stale  bread,  damp  leather,  gloves,  etc. 

beneficent  while  others  are  very  injurious  to  health.  The  yeast  organisms 
(Saccharomyces)  cause  the  alcoholic  fermentations  in  saccharine  solutions. 
Many  of  the  mould  group  cause  skin  and  other  diseases.  They  all  belong 
to  the  plant  division  fungi.  The  more  important  species  may  be  grouped 
under  three  orders,  as  follows: 

142 


YEASTS   AND    MOULDS. 


143 


I.  Phycomycetes.     (Zygomycetes.)     Zygospore  formation. 

1.  Mucor  corymbifer.  \  .      .         .  f      . 

}   Both  are  found  in  tissue  infections. 

2.  Mucor  mucedo.       } 

3.  Other  species  of  Mucor  are  reported  as  causing  pathologic  conditions 
in  man  and  in  lower  animals.     Some  are  the  cause  of  fatal  infectious  dis- 
eases in  such  household  pests  as  the  common  fly.     Others  attack  fruits, 
as  pears,  figs  in  particular,  leather  goods  as  gloves,  etc. 


FIG.  64. — Saccharomyces  cerevisea.     The  form  or  variety  known  as  brewers'  top  yeast. 

(Oberhef.) 


II.  Ascomycetes.     Spores  formed  in  asci  (sacs). 
i.  Saccharomycetes — yeasts  proper. 

a.  Saccharomyces  cerevisece.     This  name  is  applied  to  many  species 
or  varieties  of  yeasts  concerned  in  fermentation  processes,  as  in 
beer,  wine  and  sake  making. 

b.  Saccharomyces  angina.     Pathogenic. 

c.  Saccharomyces  ellipsoides.     Common  in  fermenting  fruits,  jams, 
jellies,    fruit  juices,   etc.     Other  species  are  active  in  various 
vegetable  food  fermentations. 

d.  Saccharomyces  blanchardi.     Pathogenic. 

e.  Endomyces  albicans.     Pathogenic,  causes  thrush. 


144 


PHARMACEUTICAL    BACTERIOLOGY. 


Pathogenic;  general  infections. 
Pathogenic. 


f .  Cryptococcus  gilchristi. 
g    Cryptococcus  hominis. 
Gymnoascomycetes. 

a.  Trichophyton    tonsurans.     Pathogenic,     causes    scalp     disease 
(ringworm),  also  attacks  other  external  tissues. 

b.  Trichophyton  sabourandi.     Pathogenic.     Attacks  scalp  and  beard 
(ringworm) . 


FIG.  65. — Saccharomyces  cerevisea.  The  form  or  variety  known  as  brewers'  bottom 
yeast.  (JJnterheje).  a,  Spore  formation;  b,  elongated  cells,  which  develop  under  certain 
conditions  of  moisture,  food  supply,  etc. 

c.  Trichophyton  violaceum.     Pathogenic.     Like  (b).     Violet  color. 

d.  Trichophyton  mentagrophytes.     Pathogenic.     Causes  beard  and 
body  ringworm. 

e.  Trichophyton  cruris.     Pathogenic. 

f.  Microspomm  audouini.     Pathogenic. 

g.  Achorion  schcenleini.     Pathogenic.     Is  the  cause  of  that  very 
common  scalp  disease  of  children  known  as  favus. 

3.  Carpoascomycetes. 

a.  Penicillium  crustaceum.     This  is  a  blue-green  mould  which  is 


YEASTS  AND   MOULDS. 


145 


believed  to  be  pathogenic  in  chronic  catarrhal  conditions  of  the 
Eustachian  tubes  and  of  the  stomach. 

b.  Penicillium  glaucum.     This  is  the  omnipresent  blue-green  mould 
so  common  in  the  household,  infesting  all  exposed  moist  organic 
substances.     Supposed   to  be  non-pathogenic,   although  some 
credit  it  with  being  the  cause  of  pellagra. 
Aspergillusfumigatus.     Said  to  be  the  cause  of  pellagra. 


c. 


d.  Aspergillus  concentricus.     Causes  ringworm.     Common  in  the 


FIG.  66. — Saccharomyces  ellipsoides.  Very  common  in  fruit  products  as  jams,  jellies, 
etc.  Living  yeast  cells  show  budding  of  cells  and  vacuoles.  Dead  yeast  cells  usually 
occur  singly,  the  vacuoles  are  wanting  and  the  cell  walls  are  more  distinct,  generally  due 
to  the  absorption  of  coloring  substances  in  the  medium  in  which  they  occur. 

Malay  peninsula,  China  and  in  the  Philippines.     Limited  to 
tropical  countries. 

d.  Aspergillus  flavus.    Pathogenic.     Found  in  chronic  discharges 
from  ear. 

e.  Aspergillus  repens.     Much  as  (d). 

f.  Aspergillus   pictor.     Pathogenic.     Occurs   in   Central  America, 
where  it  causes  a  mange  disease. 

g.  Aspergillus  oryzcz.     Nonpathogenic.     Cultures  of  this  fungus 
are  used  in  the  manufacture  of  sake  (Chinese  and  Japanese 
rice  wine).     The  fungus  growing  and  feeding  upon  the  steamed 
rice  grains  converts  the  starch  into  saccharine  substances  which 
are  then  acted  upon  by  the  yeast  ferment. 


146 


PHARMACEUTICAL    BACTERIOLOGY. 


III.  Hyphomycetes.     Systematic    position    of    the    pathogenic    members 
not  well  defined.     Life  history  not  yet  fully  worked  out. 

i.  Discomyces  bovis.  (Actinomyces) .  The  so-called  ray  fungus 
which  causes  the  condition  in  cattle  known  as  actinomycosis,  a  disease 
which  can  be  transmitted  to  man. 

2.  Discomyces  madurce.  (Mycetoma).  Causes  the  cattle  disease  known 
as  madura  foot,  which  can  be  transmitted  to  man.  Essentially  a  tropical 
disease.  Two  varieties  (black  and  white)  of  the  disease  are  reported. 

3.  M alas sezia  furfur.     This  is  the  fungus  which  causes  a  skin  disease 


FIG.  67. — Three  terminal  hyphae  showing  the  characteristic  spore  formation  of  Peni- 
cillium  glaucum.  This  fungus  is  a  true  saprophyte  and  is  never  found  on  living  fruits  or 
vegetables.  Mouldy  food  substances  are  quite  universally  rejected  as  being  unfit  for 
human  consumption. 

(Tinea  versicolor)  which  is  quite  common  in  tropical  as  well  as  in  temperate 
climates. 

4.  Microsporoides   minutissimus.     Causes   a    skin   disease   known   as 
Erythrasma  or  Dhobie's  itch.     Found  in  the  tropics. 

5.  Trichosporum    giganteum.     Causes    a    disease    of    the    hair.     The 
spores  of  the  fungus  are  arranged  about  the  hair  in  a  peculiar  mosaic. 


YEASTS  AND   MOULDS. 


147 


Moulds  differ  from  bacteria  in  that  they  thrive  best  in  acid  media  and  in 
that  they  are  not  so  readily  killed  by  means  of  the  usual  chemical  disinfec- 
tants. Heat  (dry  as  well  as  moist)  kills  the  hyphal  structure  quite  readily, 
but  the  spores  are  quite  resisting,  though  less  so  than  the  spores  of  bacteria. 
They  can  be  cultured  on  potato,  on  bread,  or  on  other  organic  food  materials 
(kept  moist  in  a  moist  chamber) .  The  following  medium  is  very  satisfactory. 

Maltase 4  gm. 

Peptone i  gm. 

Agar    1.5  gm. 

Water  .  100  c.c. 


FIG.  68. — Actinomyces  bovis.  Showing  the  hyphal  structure  of  this  pathogenic  fungus. 
There  are  numerous  fungi  of  the  mould  group  that  cause  local  pathologic  conditions  of  the 
skin  and  mucous  membranes. 


Mix,  dissolve,  filter,  titrate  to  reaction  +  2  and  sterilize  in  the  usual  way. 
Culturing  is  usually  done  in  Erlenmeyer  flasks  (250  or  500  c.c.)  with  a  thin 
layer  of  the  medium  in  the  bottom.  Before  placing  the  mould  material 
in  the  flask  (by  means  of  a  platinum  loop)  allow  it  to  macerate  in  60  per  cent, 
alcohol  for  two  hours  which  will  kill  the  bacteria  present  without  destroying 
the  life  of  the  mould.  The  acid  reaction  of  the  medium  (+2)  will,  however, 
usually  prevent  bacterial  growth. 


148  PHARMACEUTICAL   BACTERIOLOGY. 

Yeast  organisms  may  be  studied  very  conveniently  in  the  hanging  drop. 
The  development  of  mould  may  be  observed  between  two  sterile  slides. 
Since  these  organisms  are  much  larger  than  bacteria  there  is  little  difficulty 
in  examining  them  under  the  low  power  of  the  microscope.  Mount  in 
water  or  in  a  weak  solution  (10  per  cent.)  of  caustic  potash  or  soda.  In 
looking  for  yeasts  and  moulds  in  liquids,  centrifugalizing  may  be  desirable. 
Staining  methods  will  rarely  be  necessary. 

While  it  is  true  that  not  all  moulds  are  pathogenic,  yet  it  must  be  remem- 
bered that  many  are  decidedly  so,  besides  most  of  them  are  very  objection- 
able on  account  of  the  disagreeable  mouldy  odor  and  taste,  if  for  no  other 
reason.  Mouldy  food  substances  are  not  fit  for  consumption  and  moulds 
should  not  occur  in  any  of  the  pharmaceutical s,  syrups,  soda  fountain 
preparations  and  fruit  juices.  Most  of  the  yeasts  are  non-pathogenic.  The 
common  yeast  has  even  been  used  as  an  intestinal  disinfectant  in  typhoid 
fever,  yet  no  preparations  in  the  drug  store  should  be  allowed  to  undergo 
yeast  fermentation  for  the  reason  that  the  process  changes  the  quality  and 
flavor  of  the  substances  thus  attacked.  Fruit  pulp,  fruit  juices  and  syrups 
of  all  kinds  are  peculiarly  liable  to  the  attacks  of  the  yeast  organisms  and 
every  precaution  should  be  taken  to  guard  against  such  infection.  This  is 
not  a  simple  matter  because  the  yeast  cells  and  the  yeast  spores  are  found 
everywhere  and  develop  very  readily  in  all  saccharine,  slightly  acid  substances. 
Moist  heat  sterilization  or  pasteurization  are  the  most  effectual  means  for 
preventing  yeast  fermentations. 

The  yeast  cakes  used  by  the  housewife  in  making  bread  consist  simply 
of  pure  cultures  of  Saccharomyces.  The  cakes  must  be  kept  dry  and  in  the 
cold  (ice  chest)  to  prevent  ready  decomposition.  Even  under  the  most 
favorable  conditions  they  soon  become  worthless.  As  soon  as  the  cake 
is  mixed  with  the  bread  dough  with  adequate  warmth,  the  yeast  cells 
begin  to  feed  upon  the  various  available  food  substances  present  and 
multiply  rapidly  (by  budding),  resulting  in  the  formatin  of  alcohol  and 
liberation  of  CO2  gas,  -which  latter  in  an  attempt  to  escape,  causes  the  so- 
called  rising  of  the  bread.  If  the  dough  is  not  thoroughly  mixed,  the  gas 
liberation  is  uneven  and  the  bread  will  be  unsatisfactory,  because  there  will 
be  large  cavities  in  some  parts  of  the  loaf  and  in  other  parts  the  loaf  will  be 
solid.  Bread  must  be  baked  quickly,  after  the  rising  has  reached  the 
proper  degree,  otherwise  the  loaf  will  be  flat  and  doughy.  The  housewife 
in  the  country  simply  prepares  sour  dough  cakes  which  take  the  place  of  the 
manufactured  yeast  cakes  used  in  the  city.  In  biscuit  making  the  desired 
CO 2  gas  liberation  is  brought  about  by  the  use  of  baking  soda  and  sour 
milk  or  by  means  of  baking  powder  alone. 

The  alcoholic  fermentation  in  the  manufacture  of  beer  is  caused  by  the 
several  varieties  and  forms  of  Saccharomyces  cerevisece  (Torula  cerevisece). 


YEASTS  AND   MOULDS. 


149 


In  beer  making,  the  barley  grain  is  first  acted  upon  by  the  starch  enzyme 
(diastase)  which  converts  the  starch  into  maltose  (malt)  and  the  maltose  is  in 
turn  converted  into  alcohol  by  the  Saccharomyces.  If  the  fermentation 
product  (as  grape  wine,  apple  cider,  beer,  porter,  etc.)  is  exposed  to  the  air 
for  a  time,  the  Mycoderma  aceti  enters  and  at  once  begins  to  convert  the 
alcohol  into  acetic  acid  and  we  finally  have  vinegar.  "Hard  cider"  is  simply 
apple  wine  in  which  the  acetic  acid  fermentation  has  progressed  to  an  ad- 
vanced stage. 

In  the  manufacture  of  the  Japanese  and  Chinese  rice  wine  (sake) 
the  maltose  fermentation  of  the  starch  (in  the  rice  grain)  is  brought  about 
by  the  Aspergillus  oryzce  as  already  stated.  The  process  of  beer  and  sake 
manufacture  may  be  compared  as  follows: 


BEER. 


SAKE. 


i.  Material  Used. 


Carefully  selected  barley  is  cleaned  in 
running  water,  then  macerated  in  water 
to  induce  germination.  Rice,  wheat  and 
other  cereals  may  be  added.  Hops  are 
used. 


A  good  quality  of  rice  is  thoroughly 
washed  in  cold  water,  then  softened  by  a 
steaming  process.  No  hops  used. 


2.  Diastase  Fermentation.    Malting. 


During  the  germinating  process  a  fer- 
ment or  enzyme  (diastase)  is  liberated 
which  converts  the  starch  into  saccharine 
compounds.  The  ferment  is  unorganized 
(non-living)  and  is  soluble  in  water.  The 
germinating  and  fermenting  grain  consti- 
tutes the  beer  wort. 


The  steamed  rice  is  spread  on  mats  and 
inoculated  with  the  spores  and  hyphae  of 
Aspergillus  oryza.  This  fungus  liberates 
an  enzyme  (diastase)  which  converts  the 
starch  into  saccharine  substances.  The 
enzyme  produced  by  the  fungus  is  soluble 
in  water.  Fermentation  takes  place  in"  a 
warm  room. 


3.  Alcoholic  Fermentation. 


The  beer  wort  (Bierwiirze)  is  now  ready 
to  be  acted  upon  by  the  yeast  organisms 
(Saccharomyces  cerevisece)  which  enter  from 
the  air  or  which  may  be  added  in  pure 
culture.  The  yeast  organisms  convert  the 
saccharine  substances  into  alcohol  and 
carbonic  acid  gas  (CO2). 

The  diastase  and  the  yeast  ferments  are 
both  active  during  this  process. 


The  sake  wort  (moto)  is  prepared  by  mix- 
ing the  steamed  rice  and  fungus  (A .  oryzoz) 
in  vats.  Yeast  cells  (Saccharomyces  of 
sake)  enter  from  the  air  and  cause  alcoholic 
fermentation,  converting  the  saccharine 
substances  into  alcohol  and  carbonic  acid 
gas  (C02). 

The  diastase  ferment  (produced  by  A. 
oryza)  and  the  alcoholic  ferment  (Saccha- 
romyces) are  active  during  the  entire 
process. 


PHARMACEUTICAL   BACTERIOLOGY. 


4.  Expressing,  Cooling,  Clarifying  and  Pasteurizing. 

These  processes  are  very  closely  similar  in  beer  and  sake*  brewing.  The  differences, 
if  any,  are  slight  and  pertain  to  modifications  of  methods  employed  by  different  manu- 
facturers. Preservatives,  as  salicylic  acid,  may  be  added.  Both  beverages  may  be  rein- 
forced with  alcohol.  This  is  not  generally  done  with  sake  as  the  brewers  declare  that  the 
addition  of  foreign  alcohol  destroys  the  characteristic  flavor  or  bouquet. 


FIG.  69. — Sake  making.  A,  B,  Rice  cells  entirely  filled  with  starch  granules;  C, 
rice  cells  after  steaming,  the  starch  granules  are  broken  up;  D ,  rice  starch  granules  a, 
dextrinized,  b,  normal. 

5.  Kinds  or  Brands. 


Many  different  brands  varying  in  color, 
taste,  alcoholic  percentage,  ash  percentage, 
etc.  The  alcoholic  percentage  ranges  from 
1.5  to  6.  The  ash  percentage  is  about  8. 


Different  brands  varying  in  quality. 
The  alcoholic  percentage  ranges  from  14 
to  1 8.  There  is  a  sweet  variety  (Miriri) 
and  a  white  variety  (Shiro).  Ash  percent- 
age about  3,  frequently  less. 


YEASTS  AND   MOULDS.  151 

6.  Use  and  Properties. 

A  beverage,  usually  taken  in  compara-  Usually  taken  in  small  amounts,   pro- 

tively  large  doses,  producing  a  mild  form         ducing  a  speedy,  though  transient,  form  of 
of  intoxication.  intoxication.     Taken  as  a  wine.     In  Japan 

sak6  is  usually  heated  before  drinking. 


!n-m^M^^- 

YAVV  •-•"'"    K        3 


FIG.  70. — Sake  making.  Steamed  rice  cells  (c)  attacked  by  the  hyphae  (a)  of 
Aspergillus  oryzce  which  feed  upon  the  dextrinized  rice  starch,  converting  it  into 
sacchrine  substances.  Yeast  cells  and  bacilli  are  usually  associated  with  the  hyphal 
fungus,  feeding  upon  the  saccharine  substances  formed. 


There  are  numerous  varieties  oiSaccharomyces  concerned  in  beer  brewing. 
There  are  several  kinds  of  upper  or  top  yeasts  (Kahmhefe  Oberhefe)  and  sev- 
eral kinds  of  bottom  or  lower  yeasts  (Unterhefe),  each  kind  possessing 
supposedly  special  properties.  Just  what  part  the  more  or  less  incidentally 
associated  organisms  (as  bacteria,  moulds,  and  foreign  yeasts)  may  play  in 
the  fermentation  processes  is  not  clearly  understood.  It  is  known  that  some 


152  PHARMACEUTICAL    BACTERIOLOGY. 

of  th.ese  extraneous  organisms  may  develop  to  such  an  extent  as  to  modify 
the  quality  of  the  product  completely.  Such  fermentation  diseases  are  a 
source  of  much  annoyance  to  manufacturers,  often  resulting  in  great  finan- 
cial loss,  but  this  has  also  been  the  great  stimulus  in  compelling  the  use  of 
pure  cultures  and  in  perfecting  those  methods  which  are  known  to  improve 


FiG.  71. — Sake  making.    Aspergillus  oryzce,  showing   vegetative  hyphae  (a),  and    spore- 
forming  hyphae  (b,  c,  d). 

the  keeping  qualities  of  the  articles,  whether  foods  or  drink.  Sake  in 
particular,  does  not  keep  well,  even  with  the  greatest  care  in  manufacture  and 
with  the  use  of  preservatives.  Certain  brands  of  beer,  wine,  sake,  smoking 
tobacco,  cigars,  tea,  etc.,  are  known  to  lose  their  characteristic  flavors  within 
short  periods,  due  to  the  invasion  of  some  " disease"  producing  organism. 


YEASTS  AND   MOULDS.  153 

In  many  instances  manufacturers  have  been  blamed  for  inferiority  in  the 
quality  of  fermented  products  when  in  reality  said  articles  left  the  establish- 
ment in  perfect  condition  as  far  as  quality  is  concerned,  but  w£re^subse- 
quentiy  (in  shipment,  in  storage,  etc.)  attacked  by  some  objectionable 
organism,  resulting  in  a  complete  change  of  flavor  or  bouquet. 


& 


e 


FIG.  72. — Sake  making.  A,  Dead  or  dying  yeast  cells;  B,  active  yeast  cells  which 
convert  the  saccharine  substances  formed  by  the  aspergillus  into  alcohol.  C,  D,  yeast 
cells  and  hyphae  of  aspergillus  from  the  fermenting  vats. 

The  Japanese  soya  sauce  (fermented  soya  beans,  Glycine  hispidus)  and 
miso,  a  soup  stock  of  wheat  and  soya  beans,  is  prepared  through  the  action  of 
Aspergillus  oryzce  and  A.  wentii.  The  Javanese  arrak  is  made  from  rice 
which  is  first  acted  upon  by  a  fungus  (Ragi)  in  many  respects  similar  to  A . 
oryz<z,  and  subsequently,  the  alcoholic  fermentation  is  carried  on  by  the 
Saccharomyces,  thus  the  method  of  arrak  manufacture  is  closely  similar  to 


154 


PHARMACEUTICAL   BACTERIOLOGY. 


that  of  sake.  More  generally,  however,  arrak  is  made  from  fermented  mo- 
lasses. There  are  many  other  species  of  mould,  including  the  very  common 
Penicillium  glaucum,  which  have  the  power  of  converting  starch  into  saccha- 
rine compounds  in  the  presence  of  moisture,  but  thus  far  these  are  not  used 
industrially.  An  alcoholic  drink  of  the  East  Indies  is  prepared  from  a 
starchy  root  as  follows:  A  number  of  people,  usually  girls,  sit  about  a  large 


a/? 


FIG.  73. — Showing  the  characteristic  stellate  cells  of  the  pith  of  same  reed  used  as 
filtering  material  in  clarifying  sake.  Bundles  of  the  pith  are  placed  in  the  bottom  of 
a  perforated  cask,  forming  a  layer  a  foot  or  more  in  depth;  through  this  the  sake  percolates. 
The  impurities  are  caught  in  the  intercellular  spaces  of  the  pith. 

vessel  masticating  the  roots  which  are  then  expectorated  into  the  vessel. 
The  ferment  ptyalin  of  the  saliva  converts  the  starch  into  saccharine  sub- 
stances which  is  then  acted  upon  by  the  Saccharomyces,  resulting  in  an 
alcoholic  drink  which  is  said  to  have  a  very  peculiar  flavor.  Pressed  yeast 
cakes  for  bread  making  are  prepared  as  follows: 

The  filtered  saccharine  yeast  mash  in  vats,  is  inoculated  with  pure 
cultures  of  Saccharomyces  cerevisecz.  Active  fermentation  takes  place  in  the 


YEASTS  AND   MOULDS.  155 

<» 

presence  of  pure  air  which  is  supplied  through  pipes  leading  into  the  vat. 
The  white  scum  or  foam  which  forms  is  poured  on  fine  sieves,  washed 
with  sterile  water,  and  then  centrifugalized  to  remove  most  of  the  water. 
This  partially  dry  material  is  then  pressed  into  cakes,  thoroughly  dried  at 
a  low  temperature,  and  wrapped  in  lead  foil  to  exclude  air.  Starch  is  some- 
times added  as  a  dryer,  but  this  is  no  longer  necessary  because  of  the  improved 
methods  of  manufacture.  Good  yeast  should  be  of  a  yellowish  color,  easily 
powdered  and  should  have  a  pleasant  "yeasty"  odor. 

The  so-called  Chinese  yeast,  concerned  in  various  fermentation  processes 
is  a  mixture  of  Mucor  species,  yeasts  and  bacteria.  The  following  species 
of  mucor  are  prevalent — M.  racemosus,  M.  alternans,  M.  spinosus,  M. 
circinelloides  and  M .  boidinii.  These  have  the  power  of  converting  starch 
into  saccharine  compounds,  which  are  then  acted  upon  by  the  Saccharomyces. 
Various  alcoholic  ferments  have  been  employed  in  China  and  Japan  since 
time  immemorial. 

Nuclein  is  prepared  from  yeast  and  other  vegetable  cells  and  is  very 
much  used  in  the  treatment  of  certain  diseases  due  to  pathogenic  bacteria. 
It  is  said  to  have  strong  bacteriolytic  properties  and  to  increase  phagocytosis. 


CHAPTER  X. 
PROTOZOA  IN  DISEASE. 

Certain  low  forms  of  animal  life  are  causative  of  such  diseases  as 
malaria  and  sleeping  sickness.  These  organisms  resemble  each  other  in 
that  they  are  minute,  of  simple  structure  (single  celled)  and  in  that  they 
show  active  motion  due  to  the  presence  of  flagellae  or  cilia  or  due  to  cell 
undulations.  They  are  found  in  stagnant  water  containing  decaying  vege- 
table and  animal  matter  and  in  decaying  organic  matter.  Most  of  them 
are  non-pathogenic  and  all  are  quite  readily  killed  by  means  of  heat  and  the 
common  chemical  disinfectants.  They  do  not  occur  in  pure,  fresh  well, 
spring,  or  hydrant  water.  If  present  it  is  a  sure  sign  of  vegetable  contamina- 
tion. All  liquids  intended  for  internal  use,  showing  the  presence  of  amceba, 
infusoria  and  other  protozoa  should  be  tested  bacteriologically  for  the 
presence  of  colon  bacilli,  the  typhoid  bacillus  and  other  possible  pathogenic 
organisms,  animal  as  well  as  vegetable. 

The  following  are  the  more  important  species  of  protozoa  and  the  prin- 
cipal activities  in  which  they  are  concerned: 

I.  RHIZOPODA. — These  move  by  throwing  out  slender  protoplasmic  pro- 
jections.    Silicious  coverings  may  be  present. 

1.  Entamceba  coll. — Inhabits  the  large  intestine.     Probably  harmless. 
May  be  confused  with  phagocytes. 

2.  Entamceba  histolytica. — Causes  entero-colitis  and  dysenteric  ulcera- 
tions.     It  is  also  found  in  abscesses  of  the  liver.     Occurs  in  tropical 
countries,  less  common  in  temperate  zones. 

3.  Entamceba  buccalis. — Found  in  dental  caries.     Probably  not  patho- 
genic. 

4.  Entamceba  undulans. — Occurs  in  the  intestinal  tract. 

5.  Leydenia    gemmipara. — Identity    doubtful.     Supposed    to   have    a 
causal  relationship  to  carcinomatosis  (cancer). 

II.  FLAGELLATA. — Motion  due  to  flagellae.     Some  possess  an  undulatory 
motion.     Have  been  classed  as  bacteria  (Spirillae). 

i.  Spirochceta  recurrentis  (Spirillum  obermeieri). — This  is  the  organism 
which  causes  relapsing  fever.  The  disease  is  so  designated  because 
after  apparent  complete  recovery,  one  or  more  relapses  invariably 
follow.  It  is  not  a  very  fatal  disease  (4  per  cent,  of  deaths)  and  is,  so  far, 
rare  in  the  United  States.  It  is  and  has  been  very  prevalent  in  parts  of 
Europe.  The  disease  can  be  transmitted,  by  inoculation,  to  man, 

156 


PROTOZOA   IN   DISEASE.  157 

monkeys,  mice  and  rats.  An  immunity  treatment  has  been  attempted 
with  some  success.  Most  authorities  class  the  organism  as  a  fungus 
(Spirillum). 

2.  Spirochceta  duttoni. — This   organism   is   the   primary  cause  of   the 
South  African  tick  fever  (Tete  fever),  so-called  because  the  carrier  is  a 
species  of  cattle  tick  (Ornithodoras  moubata). 

3.  Spirochceta  novyi. — Said  to  be  the  cause  of  American  relapsing  fever. 

4.  Spiroch&ta  mncenti. — Pathogenic;  causes  throat  inflammation  (Vin- 
cent's angina). 

5.  Treponema  pallidum. — The  specific  cause  of  syphilis.     Often  other 
related  organisms  are  found  associated  with  it.     This  organism  stains 
with  difficulty. 

6.  Trypanosoma  gambiense. — This  is  the  cause  of  the  dread  sleeping 
sickness  of  Africa.     The  transmitter  of  the  infection  is  the  tsetse  fly 


FIG.  74. — A,  Spirocheta  refringens;  b,  Spirocheta  pallida.     The  cause  of  syphilis. 

(Glossina  palpalis).  Investigations  have  shown  that  the  eradication 
of  the  tsetse  fly  would  also  eradicate  the  disease  (Koch),  which  has 
practically  depopulated  large  districts  in  Africa.  Related  organisms 
cause  diseases  in  horses  (surra,  dourine  and  mal  de  caderas).  There 
are  also  many  trypanosomes  of  frogs,  fish  and  birds,  but  these  are 
probably  harmless  to  man. 

Species  of  Leishmania  cause  sores  and  ulcers  (in  tropical  countries). 
Certain  tropical  Lamblia  and  Trichomonas  species  may  cause  intestinal  and 
other  disturbances. 

The  infusoria  proper  (Ciliata),  while  exceedingly  abundant  and  widely 
disseminated,  are  mostly  non-pathogenic.  The  Balantidium  coli  is  a  com- 
mon hog  parasite  which  may  also  cause  serious  dysentery  in  man. 
III.  SPOROZOA. — Commonly  designated  as  amoebae.  Have  no  cilia,  move 
by  plasmic  contraction  of  the  cell  and  reproduce  by  spores.  Of  this  group, 
the  most  important  species  is  the  Plasmodium  malaria  which  is  the  primary 


158  PHARMACEUTICAL    BACTERIOLOGY. 

cause  of  ague  or  malaria.  The  carriers  of  the  infection  are  certain  mosquitos 
(species  of  Anopheles}.  If  the  Anopheles  group  of  mosquito  could  be  exter- 
minated throughout  the  world,  malaria  would  disappear  also.  The  organism 
is  introduced  into  the  blood  by  the  sting  of  the  insect.  In  the  blood  it  un- 
dergoes certain  cycles  of  development.  The  fever  paroxysms  are  due  to  the 
sporulation  of  the  organisms  in  the  circulatory  system.  During  the  intervals 
(non-sporulation)  there  is  no  marked  febrile  disturbance.  There  are  several 
species  of  Plasmodium  causing  the  several  forms  of  malaria.  The  benign 
tertian  (P.  vivax}  has  a  cycle  of  forty-eight  hours;  the  quartan  (P.  malaria) 
has  a  cycle  of  seventy- two  hours;  and  the  malignant  tertian  (P '.  falciparum) 
has  a  cycle  of  forty-eight  hours.  In  the  latter  type  the  paroxysms  are  so  severe 
as  to  give  rise  to  a  continued  fever.  Quinine  is  fatal  to  the  Plasmodium  and 
this  remedy  should  be  given  as  a  prophylactic  and  as  a  cure. 

The  draining  of  swamps  and  other  breeding  places  for  mosquitos  has 
reduced  malaria.  The  use  of  mosquito  netting,  screens,  etc.,  has  also 
checked  this  disease.  Small  water  areas  may  be  treated  with  crude  petro- 
leum oil  which  kills  the  mosquito  larvae. 

The  primary  cause  of  yellow  fever  is  as  yet  unknown  but  it  has  been  def- 
initely determined  that  the  carrier  is  a  mosquito,  the  Stegomyia  calopus. 
Yellow  fever  is  essentially  a  tropical  disease,  though  it  may  flourish  in  tem- 
perate zones  until  checked  by  frost  which  is  so  readily  fatal  to  the  carrier,  the 
mosquito.  It  has  been  ascertained  that  the  Stegomyia  does  not  occur  far 
from  .human  habitations,  that  it  breeds  generally  in  barrels  and  cisterns 
containing  rain  water,  rather  than  in  ponds  or  larger  bodies  of  water,  more 
remote  from  habitations.  These  discoveries  have  made  possible  a  very 
effectual  campaign  against  this  dread  disease.  The  Federal  Government 
aided  by  State  and  Local  Boards  of  Health  have  insisted  on  a  discontinu- 
ance of  those  breeding  places  which  can  be  controlled  easily.  The  larger 
more  public  breeding  places  were  covered  with  crude  oil.  Screening  win- 
dows and  doors  and  sulphur  or  Pyrethrum  fumigation  of  mosquito-infested 
houses  and  rooms  was  insisted  upon  and  individuals  were  instructed  in 
methods  of  self-protection  against  the  bites  of  mosquitos.  As  a  result  the 
yellow  fever  ravages  are  now  reduced  to  a  minimum. 


CHAPTER  XI. 

DISINFECTANTS  AND  DISINFECTION.     FOOD  PRESERVATIVES. 

INSECTICIDES. 

The  pharmacist  should  be  well  informed  regarding  disinfectants  and 
their  uses  in  order  that  he  may  assist  physicians  and  health  officers  in  carry- 
ing out  sanitary  rules  and  regulations  in  which  disinfectants  play  so  important 
a  part.  The  pharmacist  should  know  how  to  disinfect  sick  rooms,  private 
homes  and  public  buildings.  He  should  in  addition  be  informed  regarding 
the  essentials  in  the  construction  of  sanitary  homes,  shops  and  stores.  He 
should  be  able  to  give  good  advice  regarding  water  supply,  sewage  disposal 
and  on  preventive  medicine  generally.  He  should  be  well  informed  regard- 
ing the  preservation  of  foods,  the  use  and  abuse  of  food  preservatives  and  on 
food  adulteration  and  should  be  prepared  to  test  foods  as  well  as  drugs  as  to 
quality  and  purity.  He  should  be  informed  regarding  the  nature  and  use 
of  insecticides  and  pest  exterminators  generally. 

Disinfectant  is  synonymous  with  germicide  and  means  any  substance, 
usually  in  the  form  of  a  liquid  or  gas,  capable  of  destroying  bacteria  and 
their  spores,  more  particularly  the  pathogenic  forms.  A  septic  substance 
is  one  contaminated  or  infected  with  pathogenic  or  otherwise  objectionable 
bacteria.  An  aseptic  substance  is  one  free  from  bacterial  infection  or  con- 
tamination, but  not  necessarily  possessed  of  disinfecting  or  even  preserving 
power.  More  broadly  speaking,  disinfectant  means  any  ponderable  or  im- 
ponderable agent  or  substance,  destructive  to  bacterial  life  and  it  is  in  this 
sense  that  the  term  is  here  used.  Preservatives  may  be  denned  as  mild 
disinfectants;  that  is,  when  used  in  larger  amounts  or  stronger  concentration, 
preservatives  become  disinfectants.  Furthermore,  the  term  preservative 
usually  applies  to  substances  added  to  foods  for  the  purpose  of  preventing  or 
retarding  microbic  infection  and  microbic  development.  It  is,  however,  also 
applied  to  other  substances.  We  speak,  for  example,  of  wood  preservatives, 
leather  preservatives,  fur  preservatives,  etc.,  meaning  thereby  substances 
which  will  prevent  certain  decomposition  or  other  destructive  changes  in 
the  articles  named,  due  to  a  variety  of  organisms  as  mould,  larvae,  insects, 
mites,  etc. 

The  chief  purpose  in  disinfection  is  to  check  and  prevent  the  spread  of 
communicable  diseases,  by  destroying  the  primary  causes  thereof,  namely,  the 
pathogenic  bacteria  or  other  disease  producing  organisms.  The  agents  or 

159 


l6o  PHARMACEUTICAL   BACTERIOLOGY. 

substances  which  have  disinfecting  powers  or  properties  are  legion.  We 
can  only  refer  to  a  few  of  the  more  important  ones,  those  which  are  commonly 
employed,  giving  the  methods  of  their  use  and  explaining  their  action. 

Disinfectants  differ  greatly  as  to  germ  destroying  powers  and  attempts 
have  been  made  from  time  to  time  to  standardize  them  or,  in  other  words, 
to  determine  their  comparative  germicidal  efficiency,  but  thus  far  no  satis- 
factory or  generally  acceptable  method  has  been  adopted.  All  methods  appear 
to  have  some  objectionable  features.  The  technic  and  principles  involved 
in  the  standardization  of  antiseptics  include  the  following: 

1.  Selecting  some  antiseptic  as  the  unit  of  comparison,  as  a  i  per  cent, 
solution  of  phenol. 

2.  As  test  objects,  definite  quantities  of  bacterial  cultures  are  used;  as 
bouillon  cultures  of  the  typhoid  bacillus,  colon  bacillus,  hay  bacillus,  etc. 
Some  experimenters .  first  air  dry  the  bacteria  before  exposing  them  to  the 
disinfectants  to  be  tested.     There  are  a  number  of  methods  known  as  the 
"silk-thread  method,"  "the  glass-rod  method,"  "the  platinum-loop  method," 
"the  spoon  method,"  and  others. 

3.  Exposing  the  bacteria  from  a  standard  culture,  for  definite  periods 
(uniform  for  the  series  of  tests)  of  time,  to  varying  strengths  of  the  disinfec- 
tants to  be  tested. 

4.  Plating  out  (in  Petri  dishes)  the  exposed  bacteria  in  order  that  the  death 
point  may  be  ascertained. 

The  results  are  expressed  numerically  by  dividing  the  strength  of  the 
disinfectant  tested  which  will  kill  a  given  organism  in  a  given  time  by  the 
strength  of  the  phenol  solution  which  under  the  same  conditions  will  kill  the 
same  organism  in  the  same  time.  To  illustrate,  we  will  suppose  that  a  1-40 
solution  of  formaldehyde  will  kill  the  pest  bacillus  in  ten  minutes  at  37°  C.  and 
that  a  i-no  solution  of  phenol  will  kill  the  same  organism  in  the  same 
length  of  time  and  at  the  same  temperature,  then  we  get  as  the  phenol  co- 
efficient of  formaldehyde,  0.36  (40/110=0.36),  which  means  that  formalinis 
only  about  one-third  as  active  as  phenol  as  far  as  the  destruction  of  the  pest 
bacillus  is  concerned.  The  phenol  coefficient  is  also  known  as  the  Rideal- 
Walker  (R-W)  coefficient,  named  after  the  English  investigators  who 
worked  out  the  details  of  the  method.  In  time  no  doubt  an  international 
standard  method  for  testing  disinfectants  will  be  adopted.  This  would  be 
of  inestimable  value  for  comparative  purposes. 

i.  Physical  and  Mechanical  Disinfectants. 

The  following  is  an  outline  of  the  physical  and  mechanical  means  of 
disinfecting. 

A.  Cleanliness. — That  is,  bacterial  cleanliness,  or  absence  of  bacteria, 


DISINFECTANTS  AND   DISINFECTION.  l6l 

brought  about  in  a  variety  of  ways.  The  liberal  use  of  pure  water  for  washing, 
bathing  and  cleansing  purposes  is  one  of  the  oldest  methods  for  getting  rid 
of  pathogenic  and  otherwise  objectionable  organisms.  It  is,  at  the^present 
time,  one  of  the  most  effectual  means  of  disinfection,  practised  by  the  house- 
wife, the  nurse,  the  physician,  in  fact  by  all  classes  and  conditions  of  peoples. 
By  bacterial  clleanliness  we  bring  about  a  dilution,  an  attenuation,  a  dis- 
semination of  objectionable  organisms  to  such  a  degree  that  bacterial 
localization  and  infection  are  greatly  retarded  or  are  made  impossible. 
Cleanliness  prevents  filth  and  dirt  accumulation. 

B.  Pure  Air. — Pure  air,  that  is  air  free  from  disease  organisms,  is  a  prime 
essential  in  preventive  medicine.  Not  only  should  the  air  we  breathe,  be 
free  from  bacterial  infection,  but  it  should  also  be  free  from  smoke,  fumes, 
noxious  gases,  soot  and  dust.  The  air  in  many  of  our  large  cities  is  often 
quite  unsuitable  for  breathing  purposes  due  to  fumes,  soot  and  smoke  from 
numerous  furnaces  and  factorks,  stenches  from  sewage,  from  stock  yards, 
from  gas  factories,  etc.  This  should  not  be.  Stock  yards,  glue  factories,  etc., 
should  be  sufficiently  remote  from  cities  to  avoid  permeating  the  city  with 
the  horrible  stenches  emanating  therefrom.  Smoke,  fumes  and  noxious 
gases  should  not  be  permitted  to  'escape.  The  recent  tests  with  smoke  con- 
sumers, with  the  precipitation  of  fumes  and  smoke  by  means  of  electricity,  etc., 
would  indicate  that  it  is  possible  to  prevent  the  pollution  of  the  atmosphere 
by  the  above  agents.  Just  as  soon  as  there  is  a  smoke  consumer  on  the 
market  that  is  a  practical  success,  every  smoke  producing  furnace  should 
be  supplied  with  one,  irrespective  of  cost.  The  same  should  apply  to  the 
use  of  smelter  fume  precepitators.  Streets  should  be  kept  comparatively 
clean  from  dirt  and  dust  by  means  of  sprinkling  cart  and  street  sweepers 
and  cleaners. 

The  "no  spitting"  ordinances  are  largely  a  failure  simply  because  no 
provision  is  made  to  supply  the  appurtenances  necessary  to  carry  them  out. 
It  is  not  sufficient  to  simply  put  up  a  notice  stating  that  "It  is  unlawful  to 
spit  upon  the  floor,"  but  cuspidors,  or  other  receptacles  must  be  provided 
in  sufficient  numbers,  conveniently  placed,  and  furthermore  said  receptacles 
must  be  kept  clean  and  sterilized  from  time  to  time,  otherwise  they  may 
become  the  breeding  places  and  disseminators  of  disease. 

A  most  serious  defect  in  places  of  habitation  is  the  lack  of  pure  air,  as  in 
small  bed-rooms,  in  the  Pullman  sleepers,  in  sweat  shops,  in  factories,  in 
school-rooms.  Next  to  the  crowded  sweat  shops  in  our  large  cities,  the 
lower  berth  in  the  American  Pullman  car,  is  most  unsuitable  for  human 
habitation.  Rooms  for  living  purposes,  sleeping  purposes,  for  factory  use, 
office  use,  etc.,  etc.,  should  not  only  be  large  enough,  but  there  should  be 
adequate  provision  to  renew  the  air  constantly,  no  matter  how  warm  or 
how  cold  it  may  be.  We  need  a  thorough  sanitary  supervision  of  all  building 


1 62  PHARMACEUTICAL   BACTERIOLOGY. 

construction  whether  private  home,  school,  factory,  sleeping  car,  office, 
or  street  car.  There  is  plenty  of  pure  air  and  every  individual  should  have 
an  ample  supply,  for  pure  air  is  one  of  the  most  potent  factors  in  preven- 
tive medicine. 

C.  Heat. — Heat  is  one  of  the  best  disinfectants  known.     Dry  and  moist 
heat  are  used,  both  of  which  have  been  sufficiently  treated  in  the  preceding 
chapters.     Mere  dryness  is  in  itself  a  germ  destroyer.     Microbes  require 
moisture  for  their  growth.     Most  bacteria  (vegetative  cells,  not  spores)  suc- 
cumb in  a  dry  atmosphere  in  a  comparatively  short  time,  several  hours  to 
several  days.     The  spores  may,  however,  survive  dryness  for  many  months. 

The  dry-air  temperature  usually  employed  for  germicidal  purposes, 
ranges  from  100°  C.  to  150°  C.,  acting  for  one  hour  or  longer.  A  dry  heat  of 
145°  C.  acting  for  one  hour  is  sufficient  to  kill  all  bacteria,  including  the 
spores.  Temperatures  used  for  purposes  of  disinfection  and  sterilization 
range  from  60°  C.  to  120°  C.  60°  to  75°C.  is  usually  employed  in  the  pasteuri- 
zation of  milk  and  in  sterilizing  sera,  vaccines,  certain  culture  media  (as 
egg  albumen,  blood  serum),  etc.  Moist  heat  of  100°  C.  in  the  form  of  cir- 
culating steam  vapor  is  much  used.  To  obtain  a  moist  temperature  above 
1 00°  C.,  an  autoclave  is  necessary,  or  liquids  may  be  employed  which  boil 
at  a  temperature  higher  than  100°  C.  as  cumene. 

D.  Cold. — Cold,  10°  C.  and  lower,  has  decided  aseptic  properties,  that 
is,  it  checks  bacterial  growth  and  activity  very  effectually,  as  has  already 
been  explained.     Prolonged  freezing  is,  however,  necessary  to  kill  bacteria. 
Cold  may  therefore  be  considered  a  most  excellent  check  upon  bacterial 
activity,  but  it  is  a  very  poor  germicide.     Cold  is  a  universally  recognized 
and  an  extensively  used  food  preservative,  due  to  its  checking  influence 
upon  bacterial  growth. 

E.  Agitation. — The  agitation  of  gases  and  liquids  reduces  the  bacterial 
activity  therein.     Still  waters  become  stagnant  but  running  waters  do  not, 
in  the  comparative  sense,  due  in  part  to  the  difference  in  the  oxygen  content. 
Agitating  and  churning  contaminated  liquids  checks  bacterial  development 
somewhat.    The  active  circulation  of  contaminated  air  reduces  the  number 
of  bacteria  present.     Agitation  is,  however,   not  a  satisfactory  means  of 
sterilization  and  disinfection. 

F.  Sedimentation  and  Filtration. — Sedimentation  in  sewage  waters  and 
other  contaminated  liquids,  combined  with  nitration,  is  a  very  effectual 
means  of  purification.     Precipitation  and  filtration,  aided  by  chemicals  as 
alum,  iron  sulphate,  and  other  coagulants,  are  much  employed  in  the  puri- 
fication of  water  supplies. 

G.  Free  Circulation. — Free  circulation  of  air  and  water  are  most  favor- 
able to  sanitation  because  of  the  checking  influence  upon  bacterial  activity 
and  also  because  of  the  disseminating  and  diluting  effects  upon  the  organisms 


DISINFECTION  AND   DISINFECTANTS.  163 

which  may  be  present.     Circulation  is  strictly  speaking  a  means  of  cleansing. 

Purification  of  flowing  water,  as  rivers  and  small  streams,  is  effected 
very  largely  by  oxidation  and  dilution.  The  agitated  water  take-up  oxygen 
by  absorption  which  combines  with  the  organic  particles  suspended  in  the 
water  rendering  it  unsuitable  as  food  for  bacteria.  As  the  water  flows  along 
the  bacteria  are  scattered  more  and  more.  Sedimentation  is  also  an  im- 
portant factor  in  the  destruction  of  bacteria.  Gradually  the  bacteria  settle 
to  the  bottom  of  the  stream  where  they  are  brought  in  competition  with 
other  bacteria,  protozoa,  algae,  perhaps  hyphal  fungi,  etc.,  which  tend  to 
check  and  even  entirely  inhibit  their  further  development. 

H.  Light. — Sunlight  has  most  marked  germicidal  powers,  due  in  part, 
to  the  drying  effects  produced  and  in  part  to  the  actinic  or  chemically  active 
rays  of  the  sun's  light.  Numerous  investigators  have  demonstrated  the 
germ-destroying  effects  of  the  blue  and  violet-rays  and  the  ultra  violet  end  of 
the  solar  spectrum.  Bacteria  cannot  survive  in  sunlight.  Electric  light  is 
said  to  have  the  same  effect  upon  bacterial  life  as  sunlight.  The  Jf-rays 
destroy  bacteria,  likewise  does  radium,  and  these  agents  have  been  extensively 
tested  in  the  treatment  of  skin  diseases  and  superficial  tuberculosis  as  lupus, 
and  in  cancer,  but  without  satisfactory  or  conclusive  results.  The  germ- 
destroying  effects  of  sunlight  are  not  due  to  heat  as  may  be  shown  by  the 
use  of  an  alumn  tank  which  intercepts  the  heat  rays. 

I.  Electricity. — The  electrical  current  in  itself  appears  to  be  without 
germicidal  powers,  but  electricity  is  used  to  precipitate  smelter  fumes,  and 
organic  impurities  in  water,  as  already  stated.  Electricity  is  used  to  stimu- 
late seed  germination  and  it  may  be  possible  to  utilize  electrical  discharges 
or  currents  in  the  treatment  of  communicable  diseases. 

2.  Chemical  Disinfectants. 

Chemical  disinfectants  may  be  divided  into  gaseous  (or  vaporous)  and 
liquid  (solutions).  The  liquid  disinfectants  are  superior  to  the  gaseous 
disinfectants  because  direct  contact  with  the  articles  to  be  disinfected  can  be 
brought  about,  as  in  washing,  immersing  or  mixing.  Gaseous  disinfectants 
are  effective  for  surface  sterilization,  especially  useful  for  inaccessible  rooms, 
buildings,  ships,  paintings,  books,  fabric,  etc.  Both  have  their  special 
advantages,  however. 

The  number  of  chemical  disinfectants,  variously  classed  as  gaseous, 
liquid,  patent,  proprietary,  efficient,  useful,  useless,  etc.,  etc.,  is  very  great. 
We  shall  mention  only  a  few  of  the  more  powerful  kinds.  No  reliance 
should  be  placed  in  any  patented  or  proprietary  disinfectant  until  its  value  has 
been  demonstrated  by  tests  made  by  reliable  bacteriologists,  giving  its  phenol 
coefficient.  Nor  is  this  all,  not  only  must  the  disinfectant  have  actual  germ- 


164  PHARMACEUTICAL    BACTERIOLOGY. 

destroying  powers,  but  it  must  also  be  practically  usable  and  it  must  not  be 
misrepresented  as  to  its  value  and  its  application  and  use  in  practice. 

The  resistance  of  pathogenic  germs  to  disinfectants  is  extremely  variable. 
Furthermore,  the  various  disinfectants  produce  changes  in  the  tissues  and 
substances  in  and  upon  which  they  act,  which  changes  tend  to  modify,  check 
or  inhibit  the  disinfecting  powers.  Thus  a  number  of  disinfectants  may 
have  the  same  laboratory  phenol  coefficient  and  yet  their  value  as  disinfec- 
tants in  actual  practice  is  widely  different  because  of  the  difference  in  the 
effects  produced  in  and  upon  the  substances  with  which  they  are  brought  in 
contact. 

As  a  rule,  the  action  and  use  of  disinfectants  is  variable  according  to  the 
following  conditions: 

1.  Disinfectants  are  more  active  when  warm  or  hot.     In  all  disinfections 
hot  solutions  should  be  used,  if  possible  and  if  practicable. 

2.  Gaseous  disinfectants  act  only  in  the  presence  of  moisture,  as  will  be 
explained  under  formalin  and  sulphur  disinfection. 

3.  The  thoroughness  of  disinfection  is  directly  proportional  to  the  time 
that  the  disinfectants  are  allowed  to  act. 

4.  The  activity  of  disinfectants  is  directly  proportional  to  the  degree  of 
concentration,  though  there  are  noteworthy  exceptions.     Absolute  alcohol, 
for  example,  is  of  very  little  value  as  a  disinfectant,  whereas  the  weaker 
solutions  (40  to  70  per  cent.)  are  a  very  active  germ  destroyer.     The  same  is 
true  of  ether,  chloroform,  glycerin  and  a  number  of  other  substances.     Most 
disinfectants  have  a  concentration  of  optimum  or  maximum  efficiency  which 
is  the  degree  of  concentration  generally  employed  in  practice. 

5.  In  actual  practice  the  cost  of  disinfectants  is  a  very  important  de- 
sideratum, as  is  indicated  by  the  following  table  giving  the  comparative 
phenol  coefficient  and  the  relative  cost: 


Name  of  Antiseptic 

Phenol 
Coefficient 

Comparative 
Cost 

i 

Carbolic  acid 

I    OO 

• 

$          O  .  2Z 

Chinosol  (potassium  salt) 

o  o^? 

127    8? 

Condy's  fluid  (permanganates) 

o  oo 

2    OO 

Cyllin  (a  cresol)      

ii  .00 

0.08 

Formaldehyde                                 •        

O.  T.O 

4.40 

Izal  (rich  in  cresols)                                             .  .  • 

8  oo 

O.  12 

Listerine  (a  compound) 

o  03 

•724    62 

Lysoform  (formalin  soap) 

O    IO 

36    4O 

Pearson's  antiseptic  (a  cresol)      

I  .40 

0.42 

Sanitas  (contains  oil  of  turpentine)  

O.O2 

42.56 

DISINFECTANTS  AND   DISINFECTION.  165 

6.  It  is  known  that  the  disinfecting  power  of  metallic  salts  is  proportionate 
to  their  electric  dissociation,  that  is,  the  more  strongly  a  salt  is  dissociated  by 
electrolysis  the  stronger  is  its  disinfecting  power.     It  follows  that  anything 
which  interferes  with  the  electrolytical  dissociation  of  germicides  weakens 
the  germicidat  power.     For  example,  the  addition  of  sodium  chloride  lowers 
the  germ  destroying  powers  of  corrosive  sublimate  through  such  interference. 
This  is  a  matter  of  great  importance  in  determining  the  value  of  antiseptics. 

7.  The  chemical  composition  of  the  material  associated  with  the  germs 
to  be  destroyed  has  a  marked  influence  upon  the  action  of  the  germicides. 
Thus  germicides  give  different  results  when  acting  upon  the  same  organism  in 
water,  in  beef  broth,  in  salt  solutions,  in  and  upon  tissues,  etc.     For  this 
reason  the  value  of  germicides  in  actual  practice  cannot  be  based  exactly 
upon  the  laboratory  results. 

8.  Not  only  do  different  species  of  disease  germs  differ  in  resistance  to 
germicides,  but  the  different  strains  of  the  same  species  react  differently  with 
the  same  germicide.     Certain  organisms  appear  to  have  an  elective  affinity 
for  certain  chemicals,  as  for  example,  the  malaria  germ  for  quinine,  and  the 
syphilis  germ  for  mercury  salts. 

Disinfectants  destroy  or  kill  germs  in  different  ways.  In  some  cases 
the  death  of  the  organism  is  due  to  oxidation  as  when  ozone,  hydrogen 
peroxide  and  sulphites  are  used,  or  death  may  be  due  to  interference  with 
nutrition,  but  more  generally  it  is  due  to  the  coagulation  of  albumen  and 
abstraction  of  water  from  the  cell-plasm,  as  in  the  use  of  dry  heat,  phenol, 
alcohol,  tannic  acid  and  metallic  salts.  As  already  explained  in  another 
chapter,  lysins  act  by  actually  disintegrating  the  bacterial  cells. 

The  action  of  disinfectants  depends  upon  a  great  variety  of  conditions, 
entirely  too  numerous  and  too  complicated  to  be  fully  treated  in  a  text-book, 
nor  is  it  necessary  to  enter  into  lengthy  discussions  and  explanations.  In 
time  many  of  the  points  still  in  dispute  will  be  definitely  worked  out,  in  the 
laboratory. 

As  a  rule  germicides  are  most  active  when  dissolved  in  water,  though 
some  authorities  declare  that  bichoride  of  mercury,  phenol,  thymol  and  lysol 
are  more  active  when  dissolved  in  50  per  cent,  alcohol.  The  Activity  Nof 
phenol  as  a  germicide  is  greatly  increased  by  the  addition  of  hydrochloric 
acid,  whereas  lime  reduces  its  potency.  Solutions  of  germicides  in  oils 
are  inert  because  oil  does  not  penetrate  the  bacterial  cell;  however,  the  oil 
itself  may  be  fatal  to  bacterial  life,  in  which  case  the  added  germicide  is  un- 
necessary. Chemical  germicides  do,  however,  increase  the  potency  of  the 
volatile  coal-tar  products  as  gasoline,  benzine  and  xylol,  provided  the 
germicides  are  soluble  in  these  substances. 

The  following  are  the  more  important  disinfectants  given  approximately 
in  the  order  of  their  usefulness  and  potency. 


1 66  PHARMACEUTICAL    BACTERIOLOGY. 

A.  Carbolic  Acid  (Phenol). — Very  widely  used,  in  strengths  of  from  i  to  5 
per  cent.     As  a  disinfecting  wash  for  all  manner  of  septic  things,  a  5  per  cent, 
solution  is  commonly  employed.     A  2.5  per  cent,   (also  the  5  per  cent.) 
solution  is  much  used  as  a  disinfectant  for  hands  and  the  skin  generally 
and  for  septic  wound  irrigation.     A  0.5  to  i  per  cent,  solution  is  used  as  a 
mouth  wash  and  gargle.     Phenol  does  not  kill  spores  hence  should  not  be  used 
after  anthrax,  tetanus,  malignant  edema  and  other  diseases  due  to  spore 
bearing  bacteria.     Phenol  coagulates  albumen,  but  not  as  actively  as  does 
corrosive  sublimate. 

Carbolic  acid  (5  per  cent.)  is  much  used  for  disinfecting  liquid  discharges 
in  dysentery,  typhoid,  cholera,  and  for  the  disinfection  of  sputa  and  expecto- 
rations in  tuberculosis,  in  pneumonia,  etc.,  using  about  two  times  as  much 
of  the  disinfectant  as  material  to  be  disinfected,  allowing  the  mixture  to 
stand  for  several  hours  at  least. 

A  5  per  cent,  solution  may  be  prepared  as  follows: 

Carbolic  acid  (95  per  cent.),  6  1/2  oz. 

Water,  i  gal. 

Shake  thoroughly  until  all  of  the  acid  is  dissolved. 

Carbolic  acid  does  not  destroy,  bleach  or  discolor  cloth  fabric,  does  not 
corrode  metal,  has  a  marked  characteristic  odor,  is  a  powerful  escharotic 
poison,  and  the  crystals  are  readily  liquefied  by  heat,  by  alcohol  and  by  water. 

B.  Liquor  Cresolis  Compositus  U.  S.  P. — This  most  efficient  germicide 
is  a  liquid  soap  with  50  per  cent,  cresol,  miscible  in  all  proportions  with 
water.    The  cresols  used  should  have  a  high  boiling-point  (187°  to  189°  C.). 
The  germicidal  powers  of  this  substance  is  nearly  double  that  of  carbolic 
acid.     It  does  not  coagulate  albuminous  matter  and  kills  spores. 

There  are  a  number  of  germicides  similar  to  carbolic  acid  having  marked 
germicidal  properties  including  creolin,  cresol  and  lysol.  These  are  some- 
what superior  to  carbolic  acid.  Lysol  is  a  cresol  mixed  with  soap  which 
greatly  facilitates  the  solution  of  the  cresol,  being  therefore  similar  to  liq. 
cres.  comp.  U.  S.  P.  They  all  kill  spores. 

C.  Tricresol. — Tricresol  is  a  mixture  of  orthocresol,  metacresol  and  para- 
cresol.     It  dissolves  in  water  in  the  proportion  of  2.5  per  cent,  and  is  about 
three  times  as  active  as  carbolic  acid.     It  is   less  irritating  than  carbolic 
acid  for  which  reason  it  is  preferred  in  sterilizing  sera  (about  0.25  per  cent.) 
and  other  solutions  intended  for  hypodermic  use.     Tricresol  kills  spores  and 
albuminous  matter  does  not  interfere  with  its  action. 

Tricresol,  cresol,  lysol,  solveol,  solutol  and  creolin  are  usually  employed 
(as  germicides)  in  i  per  cent,  solutions  and  are  generally  conceded  to  be 
equal  to  about  2.5  per  cent,  solutions  of  phenol.  They,  however,  have  no 
superiority  over  the  liq.  cres.  comp.  U.  S.  P. 

D.  Formalin. — The  40  per  cent,  commercial   article  is  used.     It  has 


DISINFECTANTS  AND   DISINFECTION.  167 

many  advantages  as  a  disinfectant.  It  does  not  injure,  fade  or  decolorize 
cloth  or  other  colored  fabric  and  does  not  corrode  metal  (excepting  hot  steel 
and  iron).  It  kills  spores  and  is  an  efficient  deodorant.  Albuminous 
matter  does  not  interfere  with  its  action  and  hence  it  is  an  efficient  sick-room 
disinfectant.  It  disinfects  and  deodorizes  all  discharges  from  patients 
very  quickly  and  completely,  when  used  in  4  to  5  per  cent,  solutions. 

As  a  gaseous  disinfectant  it  is  active  in  a  moist,  warm  atmosphere.  It 
does,  however,  not  kill  insects  and  other  higher  organisms  and  in  this 
regard  it  is  inferior  to  sulphur  dioxide,  but  has  the  advantages  of  not  decoloriz- 
ing fabric  and  being  a  better  deodorant.  There  are  several  proprietary  dis- 
infectants composed  of  soap  and  formalin,  as  lysoform. 

E.  Sulphur. — Sulphur  in  itself  is  odorless,  tasteless  and  wholly  inert  as  a 
germicide,  but  when  undergoing  oxidation  into  sulphur  dioxide  (combustion), 
in  the  presence  of  moisture,  it  is  a  very  active  disinfectant  and  is  at  the  same 
time  fatal  to  insects  and  in  fact  to  all  forms  of  animal  life,  including  rats, 
mice,  etc.     But  it  cannot  be  used  to  disinfect  fine  fabrics,  paintings,  books, 
etc.,  because  of  the  destructive  effects  upon  pigments. 

Under  ordinary  conditions  the  gaseous  substances,  as  formaldehyde 
(formalin)  and  sulphur  dioxide,  are  surface  disinfectants  only  and  are  used 
where  surface  disinfection  is  all  that  is  required,  as  in  the  sterilization  of 
clothing,  wood  work,  walls,  ceilings,  pictures,  furniture,  etc. 

F.  Bichloride  of  Mercury. — This  is  the  most  potent  and  most  extensively 
used  of  all  antiseptics.     A  i-iooo  aqueous  solution  (used  hot  whenever  and 
wherever  possible)  makes  a  most  satisfactory  germicidal  wash  for  floors, 
walls,  wood  work  of  all  kinds,  in  fact  anything  requiring  disinfection,  ex- 
cepting metals  which  would  be  corroded  (excepting  of  course  platinum, 
gold,  silver)  and  substances  rich  in  albuminous  matter  as  pus,  sputum,  and 
other  sick  room  discharges,  which  are  coagulated  by  this  germicide,  checking 
further  action. 

The  i-iooo  solution  is  sufficiently  powerful  to  kill  all  non-sporogenous 
bacteria  at  the  ordinary  room  temperature  in  one-half  hour.  For  spores  a 
stronger  solution  (1-500)  and  longer  exposure  are  desirable  (one  hour). 

The  chief  disadvantages  to  the  use  of  corrosive  sublimate  are  its  highly 
toxic  nature,  its  corroding  effect  upon  metals  and  its  coagulating  effects  upon 
albumen  which  hinders  penetration.  It  should  also  be  borne  in  mind  that 
soap  interferes  with  the  action  of  corrosive  sublimate. 

A  i-iooo  solution  is  made  as  follows: 

Bichloride  of  mercury,  61  1/2  grs. 

Citric  acid  or  salt,  61  1/2  grs. 

Water,  i          gal. 

The  salt  or  citric  acid  is  added  to  retard  the  decomposition  of  the  bi- 
chloride. Tablets  are  now  on  the  market  prepared  from  mercury  cyanide. 


168  PHARMACEUTICAL   BACTERIOLOGY. 

They  are  held  to  be  more  decidedly  antiseptic  than  either  the  iodide  or  the 
bichlorid  of  mercury,  and  are  so  prepared  that  one  tablet  added  to  a  pint 
of  water  will  make  a  strength  of  i-iooo. 

G.  Chlorinated  Lime. — Also  known  as  chloride  of  lime.  This  is  an 
oxidizing  disinfectant  and  deodorant,  most  extensively  employed  for  the 
disinfection  of  stools,  urine,  sputa  and  other  excreta.  Eight  ounces  of  the 
chlorinated  lime  are  added  to  one  gallon  of  water.  This  solution  is  placed 
in  the  vessel  which  is  to  receive  the  discharges,  using  at  least  double  the 
amount  to  be  disinfected  and  allowing  the  mixture  to  stand  for  one-half 
hour  or  longer.  Chlorinated  lime  destroys  color  and  corrodes  all  textile 
fabrics  and  most  metals.  It  must  be  kept  in  an  air  tight  receptacle  as  it  loses 
in  strength  on  exposure  to  air.  The  solutions  should  be  made  as  required. 

H.  Lime. — Lime  (unslaked  lime,  quicklime)  is  very  useful  for  the  de- 
structive disinfection  of  cadavers  dead  of  infectious  diseases,  using  twice  the 
amount  of  lime,  by  weight,  to  the  substance  to  be  disinfected.  The  lime  is 
powdered  or  crushed  and  packed  about  the  cadaver  in  a  box  or  coffin.  Nei- 
ther water  nor  moisture  need  be  added. 

I.  Milk  of  Lime. — Lime  is  slaked  in  the  usual  way.  From  the  slaked 
lime  the  milk  of  lime  is  prepared  by  adding  eight  parts  of  water.  The 
preparation  should  always  be  made  from  freshly  slaked  lime.  It  is  much 
used  for  the  disinfection  of  stools  and  sputum,  using  an  amount  equal  to  the 
amount  of  material  to  be  disinfected.  Whitewash  is  much  used  to  disinfect 
and  preserve  fences,  stables,  sheds,  walls,  ceilings,  etc. 

J.  Copper  Sulphate. — Blue  vitriol  is  a  very  useful  disinfectant  for  sick 
room  excreta  of  all  kinds,  using  a  5  or  10  per  cent,  solution,  bulk  equal  to  bulk 
of  material  to  be  disinfected,  stirring  and  mixing  and  allowing  to  stand  for  3  to 
4  hours.  Iron  sulphate  (copperas)  is  similarly  used,  though  it  is  somewhat 
weaker  in  action. 

K.  Permanganate  of  Potassium. — This  is  another  of  the  oxidizing  anti- 
septics, having  a  rather  limited  use.  It  is  furthermore  comparatively  ex- 
pensive. Freshly  prepared  solutions  are  used,  ranging  in  strength  from 
i-iooo  up  to  5  per  cent.  Quite  extensively  used  as  a  disinfectant  for  hands. 
Has  been  administered  internally  to  oxidize  alkaloidal  poisons  in  the  stomach 
and  in  the  intestinal  tract. 

The  following  antiseptics  are  used  more  or  less  in  surgery  and  as  skin  and 
other  tissue  disinfectants.  Some  of  them  are  used  as  general  disinfectants, 
but  as  a  rule  they  are  not  sufficiently  powerful  to  be  of  much  practical  value. 

A.  lodoform. — Formerly  much  used  as  a  dressing  for  syphilitic  ulcers. 
It  is  not  germicidal  but  has  decided  aseptic  and  sedative  properties  hence 
also  used  in  scalds  and  burns.  It  may,  however,  cause  dermatitis.  It  is 
insoluble  in  water  but  freely  soluble  in  ether  and  alcohol.  The  ointment 
(containing  10  per  cent,  iodoform)  is  still  much  used.  Aristol,  europhen 


DISINFECTANTS  AND    DISINFECTION.  169 

iodol,  losophen  and  nosophen  are  iodoform  derivatives,  have  similar  proper- 
ties, less  odorous,  less  irritating  and  less  poisonous.  The  persistent  dis- 
agreeable odor  of  iodoform  is  a  great  objection  to  its  use. 

B.  Boric  Acid  and  Borax. — Boric  acid  is  a  very  mild  antiseptic  and  hence 
is  of  little  or  no  practical  value  as  a  germicide  but  it  is  an  ideal  aseptic  agent. 
It  can  be  applied  to  comparatively  aseptic  cuts,  bruises,  wounds,  etc.,  in 
saturated  solution  (aqueous)  or  in  powder.     It  can  be  applied  as  a  dusting 
powder  to  many  conditions  where  an  aseptic  substance  is  indicated.      In 
saturated  solution  it  makes  a  good  gargle,  mouth  wash,  eye  wash,  etc. 

Borax  is  similarly  used  and  has  similar  properties.  The  choice  between 
the  two  is  decided  by  the  difference  in  reaction.  Boric  acid  is  slightly  acid 
in  reaction,  whereas  borax  is  slightly  alkaline.  The  preparation  boro- 
glycerin  is  much  used  as  a  dressing  for  inflamed  and  infected  mucous 
membranes. 

Sixteen  grains  of  salicylic  acid  and  96  grains  of  boric  acid  dissolved  in  a 
pint  of  sterile  water  makes  Thiersh's  fluid.  This  is  useful  in  cleansing 
mucous  membranes,  such  as  those  of  the  mouth  and  eye,  and  it  may  be  used 
in  the  form  of  irrigations  for  cleansing  purposes. 

C.  Creosote. — This  excellent  germicide  is  rarely  used  for  general  external 
disinfection  though  it  is  more  active  than  phenol  and  does  not  coagulate 
albumen  and  is  less  toxic  and  less  irritating.     In  doses  of  from  i  to  10  minims 
(given  internally)  it  is  much  used  as  an  antiseptic  and  stimulant  in  tubercu- 
losis and  to  correct  intestinal  fermentation.     The  carbonate  of  creosote  is 
said  to  be  especially  efficacious  in  lung  troubles  (tuberculosis).     Creosote 
is  essentially  an  intestinal  antiseptic. 

D.  Hydrogen  Dioxide. — This  is  the  most  active  of  the  oxidizing  dis- 
infectants, used  in  solutions  of  from  10  to  15  per  cent.     It  is  a  very  active 
bleaching  and  deodorizing  agent.     It  is  not  used  for  general  disinfection 
but  is  one  of  the  best  known  local  germicides,  applied  to  abscesses,  ulcers, 
used  as  a  spray,  as  a  gargle,  etc.     Much  employed  in  dental  work.     Used  by 
bacteriologists  to  determine  the  amount  of  bacteria  in  milk  (indicated  by 
gas  liberation  when  added  to  the  milk  in  fermentation  tubes). 

E.  Naphthalene  Derivatives. — These  are  used  as  intestinal  antiseptics 
.but  are  of  doubtful  value  in  the  treatment  of  intestinal  diseases.     They  are 

not  acted  upon  in  the  stomach  secretions  but  on  reaching  the  intestinal  tract 
they  undergo  a  chemical  change  and  act  as  antiseptics.  Their  prolonged 
use  produces  irritation  of  intestines,  bladder  and  kidneys.  To  this  group 
belong  betanaphthol,  betol,  naphthol  naphthalin,  and  others. 

To  the  group  of  so-called  intestinal  antiseptics  belong  antipyrin,  acetan- 
ilid,  phenacetin,  phenecol,  quinine,  salicylic  acid,  salol,  salophen,  guaiacol, 
resorcin  and  many  other  substances.  Their  value  as  intestinal  antiseptics 
is  very  problematical  and  doubtful. 


170 


PHARMACEUTICAL   BACTERIOLOGY. 


F.  So-called  Respiratory  Antiseptics. — There  are  a  great  variety  of  volatile 
or  gaseous  substances  which  are  said  to  act  as  antiseptics  to  the  respiratory 
tract  when  inhaled,  as  oil  of  thyme,  eucalyptol,  oil  of  eucalyptus,  menthol, 
camphor,  euthymol,  campho-phenique,  mint  oil,  etc.,  but  their  value  in  this 
regard  is  nil.  They  may  have  some  stimulating  effect  upon  the  tissues  of 
the  respiratory  tract  but  they  do  not  destroy  any  germs  which  may  be  present 
upon  or  within  the  cells  of  the  respiratory  passages. 

The  following  table  taken  from  the  work  by  Ellis  gives  the  minimum 
proportion  of  germicidal  activity  of  well-known  disinfectants.  The  figures 
indicate  the  strength  of  solution  necessary  to  prevent  bacterial  development 
when  added  to  substances  capable  of  giving  rise  to  bacterial  growth,  naming 
therefore  the  aseptic  strength  and  not  the  actively  antiseptic  strength.  The 
figures  are  not  absolute  for  reasons  which  have  been  fully  set  forth  in  the 
beginning  of  this  chapter.  The  table  is  merely  a  guide  to  the  relative 
activity  of  the  germicides  named. 


1.  Very  active  antiseptics. 

Mercuric  iodide, 
Silver  iodide, 
Hydrogen  peroxide, 
Mercuric  chloride, 
Silver  nitrate, 

2.  Active  antiseptics. 

Osmic  acid, 

Chromic  acid, 

Chlorine, 

Iodine, 

Chloride  of  gold, 

Bichloride  of  platinum, 

Hydrocyanic  acid, 

Bromine, 

Copper  chloride, 

Thymol, 

Copper  sulphate, 

Salicylic  acid, 

3.  Fair  antiseptics 

Potassium  bichromate 
Potassium  cyanide, 
Ammonia, 
Zinc  chloride, 
Mineral  acids, 
Lead  chloride, 
Nitrate  of  cobalt, 
Carbolic  acid, 
Potassium  permanganate 


1-40000 
1-33000 

I-2OOOO 
I-I4300 
I-I25OO 


1-6666 
-5000 
-4000 
-4000 
-4000 

-3333 
-2500 
1-1666 
1-1428 
1-1340 
i-iin 

I-IOOO 


1-909 

1-909 

-714 

-526 

-500 
-500 
-500 

-333 
-285 


DISINFECTANTS   AND   DISINFECTION. 


171 


Lead  nitrate, 

Alum, 

Tannin, 

4.  Indifferent  antiseptics. 

Arsenious  acid, 
Boric  acid, 
Arsenite  of  soda, 
Hydrate  of  chloral, 
Salicylate  of  soda, 
Iron  sulphate, 
Caustic  acid, 

5.  Feeble  antiseptics. 

Calcium  chloride, 
Sodium  borate, 
Alcohol, 

6.  Very  feeble  antiseptics. 

Ammonium  chloride, 
Potassium  iodide, 
Sodium  chloride, 
Glycerin, 
Ammonium  sulphate, 


1-277 

1-222 

I-2O7 


I-I66 

-143 
-III 
-I07 
-IOO 
-  QO 
-56 


I-25 
I-I4 
I-IO 


1-9 

*-7 
1-6 
1-4 

1-4 


3.  Procedures  for  Disinfection. 

Space  will  not  permit  entering  into  a  full  discussion,  nor  is  this  necessary 
as  it  may  be  presumed  that  the  pharmacist  will  know  how  to  meet  the  special 
conditions  which  may  arise,  should  he  be  called  upon  to  do  so. 

A.  Surgical  Disinfection. — a.  The  operating  room  must  be  clean  and  free 
from  pathogenic  and  other  objectionable  organisms.  The  room  must  there- 
fore be  disinfected  from  time  to  time,  after  the  method  of  procedure  for  any 
room  which  may  be  assumed  to  be  infected,  as  will  be  explained  under  room 
and  house  disinfection.  As  to  when,  how  often  or  how  completely  the  operat- 
ing room  is  to  be  disinfected  that  must  be  left  to  the  judgment  of  the  surgeon 
in  charge. 

b.  Surgeons  should  be  especially  careful  regarding  personal  cleanliness, 
irrespective  of  the  routine  personal  disinfection  and  sterilization  performed 
preparatory  to  an  operation.     They  should  always  be  smooth-shaven  as  the 
beard  is  a  carrier  of  germs. 

c.  On  preparing  for  an  operation  the  surgeon  removes  coat,  cuffs  and 
collar  in  an  ante-room ;  rolls  up  shirt  sleeves  and  proceeds  to  wash  and  scrub 
hands  with  tincture  of  green  soap,  then  in  1-1.5  Per  cent,  tinct.  cres.  comp. 
U.  S.  P.  or  lysol,  rinse  in  sterile  water,  dry  with  a  clean  sterile  towel  and  dip 
in  50  to  60  per  cent,  alcohol.     Formalin  and  carbolic  acid  should  not  be  used 


172  PHARMACEUTICAL   BACTERIOLOGY. 

as  hand  disinfectants  (by  the  surgeon)  because  of  the  benumbing  effects  of 
these  chemicals,  causing  a  lessening  in  the  delicacy  of  touch.  A  i  per  cent, 
solution  of  potassium  permanganate  is  recommended  as  a  disinfectant  for 
hands.  In  many  hospitals  nothing  more  than  a  thorough  scrubbing  with 
green  soap  is  employed  for  the  hands  of  surgeons,  with  wholly  satisfactory 
results. 

Before  entering  the  operating  room  the  surgeon  and  attendants  don  steril- 
ized gowns  with  hoods  covering  head,  hair,  and  face  (beard),  leaving  only 
the  mouth,  nose  and  eyes  free.  The  hands  of  the  attendants  are  covered 
with  sterilized  rubber  gloves. 

d.  The  surgical  instruments  are  washed  and  wiped  dry;  boiled  for  ten 
minutes,  in  water  with  i  per  cent,  soda,  and  laid  in  a  tray  containing  5  per  cent, 
carbolic  acid  solution.  Before  using,  they  are  rinsed  in  boiled  distilled  water. 
Never  sterilize  metallic  instruments  in  corrosive  sublimate,  or  in  any  corrosive 
disinfectants  of  any  kind.  Only  a  short  exposure  would  suffice  to  dull  the 
keen  edge  of  knives,  scalpels,  and  other  cutting  instruments.  Do  not  sterilize 
steel  instruments  in  hot  air  as  high  temperatures  reduce  the  temper,  and  do 
not  sterilize  them  with  rubber  goods. 

B.  Sick  Room  Disinfection. — Disinfection  in  the  sick  room  of  a  patient 
afflicted  with  some  communicable  disease,  may  be  divided  into  disinfection 
of  dejecta,  urine  and  sputum;  disinfection  of  the  patient;  disinfection  of 
clothing  and  bedding ;  disinfection  of  the  sick  room  itself ;  and  precautionary 
disinfection  of  the  attending  physicians,  nurses  and  attendants.  In  case  of 
fatal  termination  of  the  malady  there  is  included  disinfection  after  post- 
mortem and  sterilization  of  the  dead  body.  In  all  cases,  whether  the 
patient  dies  or  recovers,  the  entire  sick  room,  including  bed,  chairs,  bedding, 
etc.,  must  be  thoroughly  disinfected.  The  methods  of  procedure  may  be 
outlined  as  follows: 

a.  Disinfection  of  Excreta. — To  disinfect  dejecta,  urine  and  sputum,  a 
4  per  cent,  solution  of  chloride  of  lime  or  a  20  per  cent,  solution  of  milk  of  lime 
will  be  found  very  efficient,  using  amounts  of  the  disinfectants  equal  to  the 
bulk  of  the  excreta  to  be  disinfected,  mixing  well  and  allowing  to  stand  for 
one  hour.  The  disinfectants  are  first  placed  in  the  vessels  intended  to  receive 
the  excreta,  more  being  added  afterward  if  it  is  thought  desirable.  If 
sputa  and  other  excreta  are  received  upon  napkins  or  other  cloth,  these  should 
be  burnt  at  once,  or  if  that  is  not  convenient  they  may  be  placed  (entirely 
immersed)  in  the  disinfectant.  For  tuberculous  sputum  the  chloride  of  lime 
is  best.  Spit  cups  should  be  kept  two-thirds  full  of  the  4  percent,  solution. 
Paper  spit  cups  are  to  be  destroyed  by  burning  as  soon  as  possible. 

Sulphate  of  copper  (5  to  10  per  cent,  solution),  or  carbo-hydrochloric 
acid  solution  (5  per  cent,  each  of  phenol  and  hydrochloric  acid)  may  be 
used  in  place  of  the  chloride  of  lime  and  milk  of  lime.  Bichloride  of  mercury 


DISINFECTANTS  AND   DISINFECTION.  173 

and  phenol  (without  the  hydrochloric  acid)  are  not  very  satisfactory  for 
disinfecting  excreta  because  of  the  coagulating  effects  upon  albuminous 
matter.  Liquor  cres.  comp.  U.  S.  P.,  lysol  and  tricresol  (2-2,5  P61"  cent- 
solutions)  may  be  used.  Weak  disinfectants  or  untried  patent  or  proprietary- 
disinfectants  should  never  be  used  for  above  purposes.  For  example, 
permanganate  of  potassium,  boric  acid,  borax,  glycothymoline,  borol,  etc., 
would  be  valueless  as  disinfectants  for  excreta. 

b.  Disinfection  of  Patient. — This  includes   cleaning   the  body  surface 
with  soap  and  water,  with  50  to  70  per  cent,  alcohol,  washing  with  i  to  2.5 
per  cent,  solutions  of  phenol,  cres.  comp.,  lysol  or  creolin,  when  so  ordered  by 
the  attending  physician.     Bichloride  of  mercury  (1-2000  to  i-iooo)  may 
be  used  for  skin  disinfection.     A  saturated  solution  of  boric  acid,  normal 
salt  solution  or  a  i-iooo  solution  of  permanganate  of  potassium  may  be  used 
as  a  wash  or  irrigation  for  non-infected  wounds  and  cuts,  etc.,  but  not  for 
ulcers,  abscesses,  etc. 

Irritating  disinfectants  should  not,  for  very  obvious  reasons,  be  used. 
In  every  case  the  mode  of  procedure  in  the  disinfection  of  the  patient  will 
be  outlined  by  the  attending  physician. 

Nurses,  attendants  and  physicians  must  observe  the  necessary  precautions 
against  becoming  disseminators  of  the  infection  and  must  resort  to  certain 
methods  of  self  disinfection  after  each  visit  to  the  patient,  as  in  small-pox, 
plague,  diphtheria  and  other  communicable  diseases. 

c.  Disinfection  of  the  Clothing  Worn  by  the  Patient  and  of  the  Bedding. — 
All  clothing  worn  by  the  patient  and  all  bedding,  as  soon  as  ordered  changed, 
should  at  once  be  immersed  in  a  hot,  5  per  cent,  solution  of  carbolic  acid  or  a 
2.5  per  cent,  solution  of  cres.  comp.  or  lysol.    After  soaking  for  several  hours 
the  clothing  should  be  boiled  in  water  for  30  minutes  at  least.    After  thorough 
drying,  preferably  in  the  sun,  the  clothing  should  be  well  ironed.     The  iron- 
ing process  in  itself  has  very  marked  germicidal  powers.     Clothing  may 
also  be  disinfected  in  formalin  (4  per  cent.) .    Sulphate  of  copper  and  sulphate 
of  iron  discolor  and  corrode  the  cloth.     All  cloth  fabrics  and  clothing  which 
has  been  in  close  contact  with  a  patient  suffering  from  diphtheria,  cholera, 
plague  or  small-pox,  should  be  destroyed  by  burning  whenever  possible. 

d.  Disinfection  of  the  Sick  Room. — The  bed  frame,  the  chairs  and  other 
wooden  furniture,    the  floor  and   the  wood   work  of   the   room,  may   be 
washed  or  wiped  with  corrosive  sublimate  (i-iooo),  formalin  (3-4  per  cent.) 
or  phenol  (5  per  cent.),  if  contamination  is  suspected  or  if  so  ordered  by 
the  physician,  even  while  the  room  is  still  occupied  by  the  patient. 

Just  as  soon  as  the  patient  is  taken  from  the  room,  a  thorough  disinfec- 
tion should  be  carried  out  at  once,  the  disinfection  including  furniture, 
clothing  of  the  patient,  bedding,  mattresses,  pillows,  etc.,  excepting  such 
articles  as  are  ordered  destroyed  by  burning. 


174  PHARMACEUTICAL   BACTERIOLOGY. 

Every  pharmacist  should  fully  inform  himself  regarding  the  state  laws 
and  city  ordinances  governing  health  and  quarantine  regulations.  State 
and  city  boards  of  health  usually  issue  free  bulletins  on  methods  of  disin- 
fection in  communicable  diseases.  Copies  of  these  should  be  on  hand  for 
ready  reference. 

For  room  disinfection,  formalin  or  sulphur  are  used.  With  formalin 
the  procedure  is  as  follows:  For  every  1000  cubic  feet  of  space  there  is 
required  one  pint  of  formaldehyde  (the  40  per  cent,  commercial  formalin) 
and  8  ounces  of  commercial  potassium  permanganate.  Place  the  perman- 
ganate in  an  agate  lined  or  iron  pail  of  about  ten  times  the  capacity  of  the  dis- 
infectant to  be  used,  spreading  the  permanganate  evenly  over  the  bottom. 
Set  pail  containing  the  crystals  upon  a  brick,  iron  stand  or  other  support,  in 
a  tub,  pan  or  dish  partially  filled  with  water.  See  that  windows  and  doors 
are  closed  and  sealed  (excepting  the  exit).  The  room  should  be  warm  and 
moist,  a  condition  which  may  be  effected  by  suspending  sheets  wrung  out  of 
hot  water  about  the  room.  In  a  steam  heated  flat,  steam  may  be  allowed  to 
escape  from  the  air  vent  of  a  radiator,  or  steam  may  be  generated  outside 
of  the  room  and  conducted  into  it  by  means  of  rubber  tubing.  Do  not 
have  an  open  fire  or  flame  in  the  room  to  be  disinfected  as  the  gas  to  be 
liberated  is  somewhat  inflammable.  Having  ascertained  that  all  is  in  readi- 
ness, pour  the  formalin  solution  from  a  dipper  or  wide  mouthed  vessel  over 
the  permanganate;  leave  the  room  at  once,  close  and  seal  exit,  plugging  key 
hole  and  crevices  in  door.  Eighty  per  cent,  of  the  gas  is  liberated  within 
ten  minutes  or  less.  Leave  the  room  sealed  for  at  least  six  hours,  preferably 
twelve  hours.  At  the  end  of  this  time  disinfection  is  complete.  Open  doors 
and  windows.  Traces  of  formalin  may  be  destroyed  by  sprinkling  or  spray- 
ing ammonia  in  the  room. 

It  is  advised  to  use  a  separate  container  for  every  pint  of  formalin  used. 
A  large  piece  of  matting  or  other  absorptive  material  may  be  placed  under 
each  container  to  guard  against  the  possibility  of  staining  the  floor,  in  case 
the  floor  requires  such  protection. 

In  case  sulphur  is  used,  prepare  the  room  (as  to  sealing,  air  moisture  and 
warmth)  as  for  formalin  disinfection,  taking  the  precaution  to  remove  (and 
disinfect  separately,  by  means  of  formalin  and  bichloride  of  mercury)  paint- 
ings, clothing  and  other  fabric  which  must  not  be  bleached  by  the  sul- 
phurous acid  fumes.  For  every  1000  cubic  feet  of  space  use  3.5  pounds 
of  flower  of  sulphur.  Place  the  sulphur  on  a  bed  of  sand  or  on  ashes  in 
an  iron  pot  or  pan  which  is  supported  on  a  brick  or  iron  stand  in  a  dish 
of  water.  Pour  a  little  alcohol  over  the  sulphur  and  ignite. 

Sulphur  candles  are  now  found  upon  the  market  and  are  more  conven- 
ient than  sulphur.  Place  a  sufficient  number  of  the  candles  upon  bricks  in 
pans  of  water  and  light  them.  Liquefied  sulphur  dioxide  put  up  in  conven- 


DISINFECTANTS  AND   DISINFECTION.  175 

lent  containers  may  be  employed,  using  15  ounces  to  each  1000  cubic  feet. 
Open  the  can  by  means  of  a  can  opener,  set  it  in  a  pan  or  dish  and  allow  the 
gas  to  evaporate. 

Remember  that  the  sulphur  dioxide  corrodes  metal,  bleaches  clothing, 
hangings  and  draperies  and,  with  formalin,  is  without  disinfecting  power 
in  the  absence  of  moisture. 

After  the  disinfection  with  formalin  or  sulphur  dioxide  is  completed, 
it  is  often  desirable  to  go  over  the  floors,  furniture,  bed  frames,  etc.,  with  a 
i-iooo  bichloride  of  mercury  solution. 

Mattresses,  heavy  quilts,  pillows  and  furniture  cushions  are  difficult  to 
disinfect  with  formalin  or  sulphur  dioxide.  These  should  be  disinfected  by 
steam  under  pressure.  In  such  diseases  as  plague,  diphtheria  and  cholera, 
such  articles  should  be  destroyed  by  burning.  Anyway,  a  sick  room  should 
have  simple  furniture  and  merely  such  articles  as  are  absolutely  necessary 
and  such  as  can  be  disinfected  readily. 

The  so-called  carbo-gasoline  method  of  book  disinfection  is  highly  rec- 
ommended. Immerse  books,  papers,  clothing  and  other  articles  to  be 
disinfected  for  twenty  minutes  in  the  carbolized  gasoline.  Take  from  the 
disinfecting  solution  and  allow  to  dry  in  the  open.  The  carbolized  gasoline 
consists  of  Baume  88°  gasoline  or  gas  machine  gasoline  to  which  2  per  cent, 
of  carbolic  acid  is  added.  No  injury  is  done  to  the  books  or  clothing, 
provided  they  are  carefully  handled  until  dry.  Gasoline  will,  however,  in- 
jure oil  paint  lettering,  etc. 

D.  Postmortem  Disinfection  and  Sterilization  of  Cadaver. — After  autopsies 
on  bodies  after  infectious  disease,  thorough  disinfection  must  be  resorted  to. 
A  liberal  use  of  a  4  per  cent,  solution  of  calcic  hypochlorite,  allowing  this  to 
act  for  at  least  one  hour,  will  serve  the  purpose. 

In  cases  of  death  from  contagious  diseases  all  orifices  of  the  body  should 
be  packed  with  cotton  well  soaked  in  a  1-500  bichloride  solution.  The 
entire  body  should  be  washed  with  a  i-iooo  bichloride  solution.  Crema- 
tion is  desirable  and  the  funeral  should  be  private. 

The  so-called  embalming  fluids  of  funeral  directors  are  aqueous  solutions 
of  various  chemical  disinfectants,  having  corrosive  sublimate  and  formalin 
as  the  chief  ingredients.  The  following  formula  is  said  to  have  the  approval 
of  the  National  Funeral  Directors  Association  of  the  United  States: 


Formalin  (40  per  cent.),  n      Ib. 

Glycerin,  4      Ib. 

Borax,  2.5  Ib. 

Boric  acid,  i      Ib. 

Potassium  nitrate,  2.5  Ib. 

Solution  of  eosin  (i  per  cent.),  i      oz. 

Water,  to  make  10      gal. 


176  PHARMACEUTICAL   BACTERIOLOGY. 

The  salts  are  dissolved  in  six  gallons  of  water;  the  glycerin,  formalin  and 
eosin  added  and  enough  water  to  make  up  the  ten  gallons. 

E.  Disinfection  of  Public  Buildings  and  Public  Conveyances. — Only  rarely 
will  it  become  necessary  to  disinfect  an  entire  large  building,  whether  private 
or  public,  and  then  the  method  of  procedure  is  much  the  same  as  for  the 
sick  room  disinfection,  already  described,  treating  each  room  as  though  it 
were  independent  of  other  rooms,  excepting  that  inner  connecting  rooms 
need  not  be  closed  and  sealed. 

In  disinfection,  one  important  fact  should  never  be  lost  sight  of,  namely, 
that  it  is  just  as  important  to  destroy  the  carriers  of  disease  (flies,  fleas,  rats, 
mice,  and  other  animals),  as  the  disease  germs  themselves.  This  is  par- 
ticularly important  in  public  disinfection,  so  much  so  that  it  is  a  general 
rule  to  always  use  a  disinfectant  which  destroys  the  disease  carriers,  as  sul- 
phur dioxide.  In  the  yellow  fever  district,  for  example,  the  chief  fumigating 
agent  is  burning  Pyrethrum  which  is  a  sure  death  to  the  Stegomyia  mosquito 
as  well  as  to  other  insects. 

Wherever  and  whenever  practical  therefore,  sulphur  dioxide  should  be 
used  for  public  disinfection.  In  many  European  cities  the  health  depart- 
ment is  provided  with  portable  generators  which  are  run  alongside  the 
building  to  be  disinfected,  the  sulphur  dioxide  generated  and  conducted 
into  the  room,  hall,  cellar,  or  area  way  to  be  disinfected,  by  means  of  tubing. 
This  is  the  safest  and  most  satisfactory  way.  If  such  apparatus  is  not 
available,  the  flower  of  sulphur,  sulphur  candles,  or  liquefied  sulphur  dioxide 
may  be  used  (15  ounces  to  each  1000  cubic  feet  of  space).  Street  cars,  rail- 
way cars,  large  public  conveyances  generally,  may  be  disinfected  much  like 
rooms,  after  being  well  sealed.  A  safe  rule  is  to  use  double  quantities  of 
the  disinfectant  for  public  conveyances,  as  compared  with  a  sick  room,  be- 
cause of  the  fact  that  it  is  difficult  to  seal  such  public  conveyances  well. 
After  the  disinfectant  has  acted  for  a  sufficient  length  of  time  (twelve  to 
twenty-four  hours),  the  place  is  opened,  aired  and  then  all  of  the  wood  work 
(of  furnishings  as  well  as  the  floor,  walls  and  ceiling)  is  either  washed  or 
sprayed  with  a  i-iooo  bichloride  of  mercury  solution  or  a  3-5  per  cent, 
formalin  solution. 

In  such  communicable  diseases  as  Have  no  animal  carriers  (other  than 
the  patient  himself)  or  where  for  obvious  reasons  such  carriers  are  not  pres- 
ent, formalin  will  always  be  the  preferred  disinfectant,  whether  for  private 
or  public  disinfection,  bearing  in  mind  that  heat  and  moisture  are  necessary 
adjuncts  to  its  use.  Formaldehyde  is  not  effective  in  a  dry,  cold  atmosphere 
because  under  those  conditions  the  formalin  is  converted  into  solid  polymer- 
ized paraformaldehyde,  which  as  such,  is  inert. 

Public  or  private  disinfection  by  means  of  formalin  may  be  carried  out 
as  follows,  the  method  selected  depending  upon  time,  place  and  opportunity. 


DISINFECTANTS  AND   DISINFECTION.  177 

a.  Wet  Blanket  Method. — Immerse  blankets  or  sheets  in  the  formalin 
solution  and  suspend  them  about  the  room  to  be  disinfected.     The  room 
may  first  be  sprayed  with  a  hot  4  per  cent,  solution  of  formalin -which  fur- 
nishes warmth  and  moisture.     The  operator  must  work  rapidly  as  formalin 
is  very  irritating  to  eyes  and  respiratory  tract. 

b.  Methyl  Alcohol  Lamps. — Formalin  may  be  generated  in  the  space  to 
be  disinfected  by  oxidizing  the  methyl  alcohol  and  converting  it  into  for- 
maldehyde.    Lamps  of  special  construction  are  necessary.     The  vapor  of 
methyl  alcohol  is  passed  over  a  highly  heated  plate  whereupon  it  is  oxidized 
into  formaldeyhde  (CH3OH+ O  =  HCHO  +  H2O)  with  liberation  of  water. 
This  method  of  disinfection  is  now  rarely  employed. 

c.  Sanitary  Construction  Company's  Lamp. — The  mechanism  consists  of 
a  tank  to  hold  the  formalin,  connected  with  a  spiral  tube  through  which  the 
solution  is  slowly  passed  through  a  flame.     The  heat  vaporizes  the  formalin 
which  is  then  conducted  into  the  room  (through  the  key  hole)  by  means  of 
suitable  tubing.     This  apparatus  is  much  used  by  health  officers. 

d.  The  Shering  Lamp. — These    small    compact   and   most   convenient 
lamps  can  be  secured  from  any  wholesale  drug  supply  house.     With  this 
apparatus  the  solid  tablets  of  paraform  or  paraformaldehyde  are  used.     The 
heat  from  the  lamp  decomposes  the  tablets,  producing  formaldehyde.     The 
lamps  are  placed  in  position,  in  sufficient  numbers,  lighted  and  the  small 
tray  of  each  lamp  is  supplied  with  a  sufficient  number  of  tablets.     As  a 
precautionary  measure  each  lamp  should  be  placed  on  a  brick  in  a  pan  or 
dish  of  water.     The  air  in  the  room  must  be  warm  and  moist. 

e.  Formaldehyde  Candles. — These  consist  of  a  mixture  of  paraformalde- 
hyde and  paraffin,  wax,  tallow  or  other  combustible,  which  may  be  moulded 
into  candles.     The  candles  are  placed  in  a  fireproof  dish  or  pan  and  ignited. 
For  room  disinfection  these  candles  are  most  convenient  as  well  as  satisfactory. 

F.  Disinfection  at  Quarantine  Stations. — All  civilized  nations  maintain 
a  system  of  vigilance  as  a  protection  against  the  introduction,  from  foreign 
countries,  of  certain  communicable  diseases  designated  as  quarantinable. 
The  first  disease  against  which  a  quarantine  was  established  was  the  plague. 
In  the  fourteenth  century  certain  Italian  cities  established  a  quarantine 
against  this  dread  disease  and  the  word  ' 'Quarantine'7  came  into  general 
use  because  of  the  fact  that  the  period  of  detention  was  about  forty  days 
(Ital.  quarantina) .  The  actual  period  of  detention  as  now  enforced  varies 
somewhat  depending  upon  the  nature  of  the  disease  against  which  the 
detention  is  maintained,  as  determined  by  the  period  of  incubation.  The 
quarantinable  diseases  recognized  by  the  United  States  are  plague  (bubonic), 
small-pox,  yellow  fever,  Asiatic  cholera,  leprosy  and  typhus.1  The  enforce- 

1  National  quarantine  against  foreign  disease  is  entirely  distinct  from  state  or  city 
quarantine.  The  following  diseases  are  recognized  as  quarantinable  by  most  state  boards. 


178  PHARMACEUTICAL   BACTERIOLOGY. 

ment  of  the  quarantine  regulations  is  under  the  direction  of  the  Public 
Health  and  Marine  Hospital  Service.  The  most  important  quarantine 
stations  in  the  United  States  are  at  San  Francisco,  New  Orleans,  New  York 
and  Boston,  ranking  in  importance  in  the  order  named.  The  Station  at 
San  Francisco  is  of  special  importance  because  upon  its  efficiency  depends 
very  largely  the  exclusion  of  plague,  cholera  and  small-pox,  the  three  highly 
communicable  diseases  so  prevalent  in  the  Orient.  Of  course  a  national 
quarantine  to  be  effective  must  be  complete,  covering  every  port  of  entry, 
whether  large  or  small,  maritime  or  inland.  This  is  very  often  not  the  case 
and  as  a  result  an  epidemic  may  enter  via  a  minor  port  where  the  service  is 
inadequate  due  to  incompetent  or  insufficient  inspection. 

.  The  quarantine  officers  are  kept  informed  as  to  the  occurrence  of  epi- 
demics or  sporadic  cases  of  quarantinable  diseases  in  foreign  countries 
and  port  cities  thus  putting  them  on  their  guard  as  to  the  need  of  special 
vigilance  regarding  imports  and  immigration  from  such  places  or  cities. 
However  every  ship  from  a  foreign  port  on  arriving  within  the  quarantine 
zone  of  the  station  is  visited  by  the  boarding  officer  who  immediately  proceeds 
to  get  data  regarding  the  sanitary  conditions  on  board,  as  to  deaths,  sick- 
ness of  any  kind,  etc.  All  passengers,  including  the  ship's  crew,  are  lined 
up  and  inspected  by  the  boarding  officer.  If  nothing  untoward  is  reported 
or  detected  the  captain  of  the  ship  is  given  a  clean  bill  of  health  and  the 
vessel  is  permitted  to  dock  and  discharge  passengers  and  cargo. 

If  however  the  boarding  officer  finds  a  case  of  small-pox  or  other  quaran- 
tinable disease  on  board,  the  ship  is  anchored  near  the  station;  the  passengers 
and  crew  are  landed  at  the  quarantine  station  and,  with  the  aid  of  the  ship's 
officers,  the  quarantine  officer  proceeds  to  disinfect  all  persons  and  their 
personal  effects,  the  same  class  distinction  (first  cabin,  second  cabin,  steerage, 
ship's  crew)  being  maintained  as  on  ship.  Each  day,  as  long  as  the  quar- 
antine lasts,  all  persons  are  examined  by  the  chief  officer  of  the  station,  to 
note,  if  possible  the  first  manifestations  of  new  cases.  Just  as  soon  as  a 
new  case  is  found  the  patient  is  at  once  taken  care  of  in  an  isolated  hospital. 
Suspects  are  kept  under  observation  in  an  isolated  camp. 

All  personal  effects,  including  every  bit  of  clothing  worn,  is  disinfected 
in  enormous  double  walled  cylinders,  by  means  of  hot  formalin  laden  steam 
under  pressure.  Sterilization  is  made  absolutely  complete  without  any 
injury  to  the  clothing. 

The  ship  with  its  cargo  is  next  disinfected  with  sulphur  dioxide  gas 
generated  in  iron  pots  or  pails  placed  upon  sheets  of  tin.  A  little  alcohol 
is  poured  over  the  sulphur,  ignited,  the  exits  closed  down  and  kept  closed 
for  twelve  hours.  If  the  cargo  contains  combustible  material  as  alcohol,  oil, 

of  health:  Scarlet  fever  (including  scarletina  and  scarlet  rash),  diphtheria  (including 
membranous  croup),  small-pox,  epidemic  cerebro-spinal  meningitis,  anterior  poliomyelitis, 
leprosy,  and  bubonic  plague. 


DISINFECTANTS  AND   DISINFECTION.  179 

benzine,  etc.,  the  sulphur  dioxide  is  generated  upon  a  special  boat  or  float 
which  is  run  alongside  and  the  fumes  conducted  into  the  hold  of  the  ship 
to  be  disinfected.  The  sulphur  fumes  kill  all  organisms  present^  including 
fleas,  rats  and  mice.  In  fact  sulphuring  of  ships  must  be  resorted  to  quite 
frequently  for  the  sole  purpose  of  killing  rats  and  mice,  even  though  there 
may  have  been  no  disease  on  board. 

4.  Purification  and  Sterilization  of  Water  Supplies. 

Every  city,  town,  hamlet  and  home  should  have  an  ample  supply  of 
pure  water  for  drinking,  cooking  and  cleansing  purposes.  Impure  waters, 
that  is  waters  which  require  sterilization  in  order  to  render  them  potable, 
are  always  dangerous.  It  is  therefore  of  prime  importance  to  secure  a  pure 
supply  of  water,  sufficiently  pure  to  make  the  work  of  sterilization  and 
purification  wholly  unnecessary;  if  that  is  not  possible,  and  it  generally  is 
not,  under  our  peculiar  communal  condition,  then  said  questionable  water 
supply  should  be  thoroughly  sterilized  and  purified,  according  to  the  most 
approved  modern  methods.  We  cannot  condemn  too  strongly  the  generally 
prevalent  methods  of  emptying  the  sewage  of  our  cities  and  towns  into 
rivers  and  lakes  and  then  again  supplying  this  sewage  contaminated  water 
to  towns  and  cities  for  drinking  and  cooking  purposes.  There  should  be 
an  efficient  state  board  of  health  cooperating  with  a  Federal  department, 
and  there  should  be  efficient  and  competent  sanitary  inspectors  to  look  after 
the  water  supplies  of  private  homes,  of  towns  and  in  the  country. 

The  suitability  of  water  for  drinking  purposes  is  inversely  proportional 
to  the  number  of  bacteria  present.  Pure  spring  or  well  water  contains 
very  few  bacteria,  rarely  exceeding  50  per  c.c.  Sewage  contaminated  water, 
which  is  still  used  for  drinking  and  cooking  purposes,  may  contain  several 
million  bacteria  per  c.c.  It  has  been  proven  time  and  again  (statistically) 
that  the  mortality  rate  (due  to  disease)  of  cities  is  practically  proportional 
to  the  purity  of  the  drinking  water  supply.  It  is  self  evident  that  water 
purification  should  be  considered  a  subject  of  the  utmost  importance.  It 
should  receive  more  attention  than  it  does. 

The  sedimentation  and  filtration  method  for  removing  dirt,  sand  and  other 
coarser  particles  from  the  water  supplies  of  large  cities  is  practised  and  has 
been  practised  for  years  in  many  of  the  European  cities.  This  is  satisfactory 
as  far  as  it  goes,  but  it  does  not  go  far  enough.  The  filtering  material  used 
(sand,  charcoal,  etc.)  does  not  remove  bacteria  and  other  small  organisms, 
excepting  those  which  are  attached  to  the  coarser  particles  remaining  upon 
the  filtering  material.  Furthermore,  unless  the  filter  is  frequently  changed 
or  sterilized,  the  filtering  material  will  become  the  breeding  place  of  germs 
and  thus  contaminate  the  water  still  more. 

Various  chemical  disinfectants  have  been  tried,  but  most  of  them  have 


l8o  PHARMACEUTICAL    BACTERIOLOGY. 

proven  unsatisfactory  for  various  reasons.  The  use  of  high  attenuations 
(1-5,000,000  to  1-50,000)  of  copper  sulphate  has  been  highly  recommended, 
especially  by  the  U.  S.  Dept.  of  Agriculture,  and  has  in  many  instances 
given  excellent  results,  especially  in  the  destruction  of  low  forms  of  algae 
and  protozoa.  As  a  means  of  destroying  bacterial  life  the  method  is, 
however,  not  a  success.  Dr.  Kraemer  and  others  recommend  the  use  of 
copper  foil  or  plates  immersed  in  the  water  as  a  means  of  destroying  patho- 
genic and  other  bacteria,  but  this  method  does  not  appear  to  have  met 
with  any  general  approval.  Kraemer  sums  up  the  copper  foil  treatment 
of  water  as  follows: 

1.  The  intestinal  bacteria,  like  colon  and  typhoid,   are  completely  de- 
stroyed by  placing  clean  copper  foil  in  the  water  containing  them. 

2.  The  effects  of  colloidal  copper  and  copper  sulphate  in  the  purifica- 
tion of  drinking  water  are  in  a  quantitative  sense  much  like  those  of  filtra- 
tion, only  the  organisms  are  completely  destroyed. 

3.  Pending  the  introduction  of  the  copper  treatment  of  water  on  a  large 
scale  the  householder  may  avail  himself  of  a  method  for  the  purifications 
of  drinking  water  by  the  use  of  strips  of  copper  foil  about  31/2  inches  square 
to  each  quart  of  water,  this  being  allowed  to  stand  over  night,  or  from  six 
to  eight  hours,  at  the  ordinary  temperature,  and  then  the  water  drawn  off 
or  the  copper  foil  removed. 

The  alum  method  of  purifying  water  has  met  with  considerable  success, 
but  more  recently  the  alum-sodium  hypochlorite  combination  has  proven 
more  satisfactory.  The  alum  coagulates  and  precipitates  the  organic  im- 
purities and  the  sodium  hypochlorite,  through  its  electric  dissociation,  acts 
as  a  germ  destroyer.  The  coagulated  and  precipitated  organic  material 
holding  most  of  the  bacteria  is  then  removed  by  filtration.  The  amount  of 
chemicals  used  depends  somewhat  upon  the  degree  of  contamination.  With 
highly  contaminated  waters  it  is  customary  to  use  3.3  per  cent,  of  alum  as 
the  coagulant,  subsequently  introducting  1.2  per  cent,  of  the  hypochlorite. 
The  water  is  then  filtered,  whereupon  it  is  ready  for  use. 

Small  quantities  of  drinking  water  may  be  purified  as  follows:  Dissolve 
a  level  teaspoonful  of  powdered  chloride  of  lime  in  a  teacup  of  water.  This 
solution  is  diluted  with  three  cupfuls  of  water,  and  a  teaspoonful  of  the 
whole  quantity  is  added  to  each  two-gallon  pail  of  drinking  water.  This  will 
give  0.4  or  0.5  part  of  free  chlorine  to  a  million  parts  of  water  and  will,  in 
ten  minutes,  destroy  all  typhoid  and  colon  bacilli  or  other  dysentery-produc- 
ing organisms  in  the  water.  Moreover,  all  traces  of  chlorine  will  disappear 
rapidly. 

There  are  in  use  a  number  of  methods  for  dissociating  sodium  hypochlor- 
ite by  electricity.  Some  of  them  are  patented  and  modifications  thereof  are 
in  use  by  city  water  purification  works,  giving  excellent  results.  Dr.  C.  P. 


DISINFECTANTS  AND   DSINFECTION.  l8l 

Hoover,  assistant  chemist  of  the  Columbus  Board  of  Health,  has  the  fol- 
lowing to  say  regarding  the  process: 

"  There  are  two  general  types  of  electrolyzers  for  dissociating  sodium 
chloride.  In  one  the  cathodic  and  anodic  products  are  allowed  to  recombine 
in  the  main  body  of  the  electroyte  and  in  the  other,  known  as  the  diaphragm 
process,  the  products  are  removed  separately  from  the  cell  as  produced. 

"For  the  production  of  sodium  hypochlorite  the  non-diaphragm  process 
has  been  considered  best  because  it  dispenses  with  the  destructible  dia- 
phragms and  the  loss  of  energy  that  all  such  diaphragms  occasion. 

"  When  a  direct  current  of  electricity  is  passed  through  a  solution  of  sodium 
chloride,  sodium  is  liberated  at  one  pole  and  chlorine  at  the  other.  The 
liberated  sodium  reacts  on  the  water  breaking  it  up  into  hydrogen  and  hy- 
droxyl  ions  to  form  sodium  hydrate.  The  sodium  hydrate  in  turn  combines 
with  the  chlorine  to  form  sodium  hypochlorite,  (Na  O  Cl)  which  becomes 
active  in  the  sterilization  of  the  water." 

Pharmacists  find  considerable  demand  for  distilled  water  for  drinking 
purposes  as  well  as  for  use  in  dispensing.  However,  some  of  the  leading 
authorities  declare  that  drinking  distilled  water  is  objectionable,  because 
of  the  disturbance  of  the  osmotic  pressure  in  the  cells  of  the  digestive  tract. 
That  is,  the  distilled  water  acts  as  a  mechanical  poison.  There  is  an  excessive 
endosmosis  inducing  an  abnormal  distention  of  the  cells,  causing  physiological 
disturbances.  This  action  is  due  to  the  fact  that  the  mineral  salts  present 
in  natural  drinking  water  are  absent  in  distilled  water. 

The  pharmacist  can  prepare  cheaply  and  simply  a  marketable  drinking 
water  which  does  not  have  the  objectionable  qualities  above  referred  to. 
Instead  of  distilling  the  water,  filter  it,  using  a  Pasteur- Chamberland  filter. 
Whether  a  large  or  small  filter  is  used  will  depend  upon  the  number  of 
customers  to  be  supplied.  In  all  probability  a  two-  or  three-tube  filter  is 
large  enough  for  the  average  retail  store.  " Rapid  safety  filters"  are  of  no 
value  whatever,  and  should  not  be  used,  as  they  are  in  no  sense  germ-proof. 
They  merely  remove  the  coarse  filth.  It  is  true  that  the  Pasteur- Chamberland 
filters  are  not  absolutely  germ-proof,  but  they  remove  most  of  the  microbes 
present,  as  may  be  determined  bacteriologically  by  the  pharmacist  himself. 
The  few  germs  which  may  pass  through  the  filter  are  killed  by  heating  the 
water  to  the  boiling-point  or  30  minutes.  Such  filtered  and  heat  sterilized 
water  should  be  sold  in  large  sterile  glass  or  earthenware  containers.  It  is 
more  palatable  than  distilled  water  and  does  not  interfere  with  the  physio- 
logical action  of  cells. 

5.  Food  Preservatives. 

The  use  of  food  preservatives  is  as  old  as  the  history  of  man.  Since 
remotest  antiquity  man  has  found  it  necessary  to  accumulate  a  supply  of  food 


182  PHARMACEUTICAL   BACTERIOLOGY. 

during  the  seasonal  periods  of  plenty  in  order  to  tide  over  the  periods  of 
scarcity.  The  very  first  observation  made  was  that  the  accumulated  and 
stored  food  soon  showed  a  tendency  to  undergo  decomposition.  The  next 
observation  no  doubt  was  that  under  certain  conditions  some  organic  food 
kept  better  than  under  other  conditions,  thus,  for  example,  primitive  man 
gradually  learned  that  sun-dried  meats  did  not  decompose  nearly  as  quickly 
as  undried  meats.  No  doubt  the  value  of  smoking  meats  was  soon  ascer- 
tained, in  all  probability  purely  accidentally,  from  meats,  etc.,  which  had 
been  exposed  to  the  smoke  of  the  camp  fire.  The  preservative  value  of  heat, 
as  in  cooking  and  roasting,  was  noted.  Next,  no  doubt  the  preservative 
properties  of  certain  chemicals  used  with  foods,  as  ashes  from  the  camp  fire, 
salt,  brine,  vinegar,  wine  (alcoholic  beverages)  and  sugar  was  noted.  Thus 
primitive  man  made  use  of  the  germicidal  powers  of  sunlight,  drying,  dry 
heat,  moist  heat,  wood  ash,  smoke,  creosote  (in  smoking  meats),  salt  solutions, 
acids  (in  vinegar)  and  alcohol,  without  having  any  idea  as  to  why 
these  agents  retarded  or  prevented  the  decomposition  of  organic  food 
substances. 

In  modern  times  the  use  of  food  preservatives  is  based  upon  th'e  germ 
theory  of  decomposition.  The  time-honored  preservatives  above  referred 
to  have  continued  in  use  and  many  new  ones  have  been  added,  as  benzoic 
acid,  sodium  benzoate,  boracic  acid,  borax,  salicylic  acid,  sodium  sulphite, 
sulphurous  acid,  formalin  and  many  others.  A  somewhat  generalized 
theoretical  assumption  is  that  the  chemical  preservatives  in  foods  are  more 
or  less  injurious  to  health.  It  cannot  be  denied  that  some  of  the  preserva- 
tives used  are  irritating  to  the  kidneys  and  skin  and  some  perhaps  interfere 
more  or  less  with  food  digestion  and  assimilation.  It  has  long  been  known, 
for  example,  that  the  prolonged  consumption  of  salted  meats  produces  serious 
skin  affections  designated  as  scurvy.  The  sulphites  are  irritating  to  the  kid- 
neys; formalin  interferes  with  digestion  of  foods,  etc.  However,  there  can  be 
little  doubt  that  in  the  comparative  sense  it  is  far  more  conducive  to  health 
and  longevity  to  eat  preserved  foods  than  foods  which  are  more  or  less 
decomposed.  We  are  daily  making  use  of  foods  which  contain  small 
quantities  of  natural  preservatives.  Cranberries,  for  example,  contain 
benzoic  acid;  formalin  and  phloroglucin  are  present  in  minute  quantities  in 
certain  plants;  a  multitudinous  variety  of  salts,  acids,  sugars,  aromatic  oils, 
etc.,  are  present  in  food  plants.  Food  chemists  do  not  appear  to  be  seriously 
worried  about  these  natural  preserving  agents  nor  about  the  old-time  artificial 
preservatives  as  smoke  creosote,  salt,  brine,  sugar,  and  vinegar,  and  it  is 
reasonable  to  suppose  that  careful  investigation  will  disclose  new  chemical 
preservatives  which  are  superior  to  those  mentioned.  The  whole  discussion 
regarding  artificially  added  chemical  food  preservatives  will  no  doubt  sim- 
mer down  to  the  following:  What  is  the  smallest  amount  of  the  least  objec~ 


DISINFECTANTS  AND   DISINFECTION.  183 

tionable  chemical  food  preservatives  which  must  be  added  to  certain  food  sub- 
stances in  order  to  preserve  them  until  they  are  to  be  consumed?  Also  the  follow- 
ing correlative  rule  should  hold  good:  No  chemical  food  preservatives 
whatsoever  should  be  used  as  such  excepting  in  cases  were  modern  methods 
of  Jieat  and  cold  sterilization  and  preservation  fail  or  are  inapplicable. 

The  use  of  sugar  and  of  salt  in  moderation  are,  of  course,  always  permis- 
sible, since  these  substances  are  essentials  in  many  foods.  The  objection 
and  danger  in  the  use  of  food  preservatives  lie  in  the  fact  that  careless  manu- 
facturers are  too  prone  to  use  them  in  order  to  avoid  employing  harmless, 
though  perhaps  less  simple,  and  more  expensive  means  of  food  preservation. 
Chemical  preservatives  make  it  possible  for  the  unscrupulous  to  use  decom- 
posed and  otherwise  objectionable  food  material.  Furthermore,  there  is  a 
strong  tendency  to  use  chemical  preservatives  in  excess,  in  spite  of  the 
strictest  legal  quantitative  limitations. 

The  following  is  a  brief  summary  of  the  more  common  food  preservatives 
and  their  use. 

The  physical  and  mechanical  means  of  food  preservation  have  been 
referred  to,  likewise  the  use  of  heat,  cold,  smoke,  etc.  One  of  the  most 
satisfactory  methods  of  preserving  foods,  now  employed  in  all  up  to  date 
canneries,  is  a  combination  of  heat  sterilization  with  air  exclusion  (air  pump 
and  by  displacement).  The  food  products  as  meat,  corn,  beans,  asparagus, 
peas,  jams,  jellies,  preserves,  etc.,  etc.,  are  heated  (100°  C.)  to  destroy  all 
germ  life,  the  containers  (tins,  glass)  are  also  heated  and  then  entirely  filled 
to  exclude  as  much  air  (oxygen)  as  possible.  Air  (oxygen)  is  necessary  for 
the  growth  of  bacteria,  yeasts  and  moulds,  hence  a  well  filled  container,  with 
a  minimum  of  oxygen  is  less  likely  to  show  decomposition  effects  (" swells," 
"leaks")  than  containers  which  are  not  well  filled.  It  is  claimed  that  whole- 
some fruit,  meat,  etc.,  (free  from  decomposition),  which  is  well  sterilized  by 
steam  heat  and  put  up  in  well  sterilized  containers  requires  no  chemical  pre- 
servative whatever.  It  is,  however,  customary,  in  the  case  of  fruits,  to  add 
sugar  as  a  preservative  and  also  for  the  purpose  of  rendering  the  article  more 
palatable.  The  sugar  from  sugar  cane  is  quite  universally  used  in  prefer- 
ence to  the  sugar  from  the  sugar  beet.  This  is  no  doubt  due  to  the  fact  that 
sugar  beet  sugar  contains  slightly  more  organic  impurities  and  is,  hence,  under 
similar  methods  of  use  as  to  quantity,  degree  of  heat  sterilization,  etc.,  slightly 
more  likely  to  undergo  decomposition. 

Preservation  of  food  substances  by  drying  is  coming  into  use  more  and 
more.  By  this  method  it  is  possible  to  keep,  for  variable  periods  of  time,  a 
great  variety  of  foods  as  apples,  peaches,  pears,  bananas,  potatoes  and  many 
other  vegetables,  besides  bread,  meats,  eggs,  milk  and  other  substances, 
which  were  formerly  more  generally  preserved  by  the  canning  method. 
Eggs  may  also  be  preserved  entire  by  giving  them  a  coating  of  tallow,  wax, 


184  PHARMACEUTICAL    BACTERIOLOGY. 

paraffin  or  soluble  silicate,  which  exclude  the  air,  or  they  may  be  preserved 
in  brine,  salt  or  other  so-called  harmless  chemical  preservative. 

Herring,  cod  and  other  fish  are  often  preserved  in  a  brine  of  salt  or  of  equal 
parts  of  salt  and  borax  or  boric  acid.  Of  meats,  fish  is  particularly  liable 
to  decomposition  and  it  is  declared  that  certain  kinds  cannot  be  preserved 
in  salt  alone,  that  it  is  necessary  to  add  boric  acid,  rubbing  the  preservative 
well  into  incisions  made  along  the  spinal  column  where  the  decomposition 
develops  earliest.  Salt  is  used  with  meats  generally  and  with  butter.  Two 
per  cent,  of  salt  in  butter  is  sufficient,  though  as  much  as  15  per  cent,  and 
more  is  sometimes  added  to  increase  the  weight.  A  combination  of  salt 
and  saltpeter  is  added  to  meat  (brine).  The  saltpeter  gives  a  red  tint  to  meat 
besides  serving  as  a  preservative.  Saltpeter  is  considered  more  or  less  in- 
jurious to  health,  when  taken  with,  food  to  the  amount  of  0.5  of  i  per  cent, 
or  more. 

Borax  and  boric  acid  is  often  added  to  milk.  4.4  grains  to  the  pint  (0.05 
per  cent.)  keeps  milk  sweet  for  a  time  (10  to  14  hours  and  longer).  Small 
doses  of  borax  and  boric  acid  (up  to  i  gram  per  day)  is  considered  harmless. 
Certain  preservatives  of  a  proprietary  nature  as  " Preserving  Salts,"  " Pre- 
servative," consist  of  borax  and  salt  in  the  proportion  of  three  to  one. 

Formalin  (the  40  per  cent,  commercial  solution)  added  to  milk,  to  the 
amount  of  1-50,000,  retards  souring  for  several  hours;  i-i 0,000  prevents 
souring  for  twelve  hours  and  longer,  and  in  this  amount  it  does  perhaps  very 
little  harm,  though  it  is  believed,  due  to  its  coagulating  effects,  to  interfere 
with  the  digestibility  of  milk,  particularly  in  children.  Several  marketed 
milk  preservatives  have  formalin  for  their  principal  ingredient  ("  milk-sweet," 
"iceline,"  "freezine"). 

Sulphurous  acid  and  sulphites  are  added  to  vinegar,  pickles,  catsups,  etc., 
anchovy  pastes,  canned  and  dried  fruits,  etc.,  to  the  amounts  of  0.2  to  1.15 
per  cent.  The  part  active  as  a  preservative  is  the  available  SO2  which  is 
gradually  oxidized  into  sulphates.  These  agents  are  deodorant,  as  well  as 
preservative,  because  of  the  high  oxidizing  power. 

Butchers  use  sulphite  preservatives  to  dust  over  sausage  meats  for  the 
double  purpose  of  giving  the  meat  a  red  color  (due  to  the  O  combining 
with  the  hemaglobin  of  the  blood)  and  to  destroy  possible  odors  of  decom- 
position. 0.05  per  cent,  of  sulphites  is  sufficient  to  check  decomposition  in 
fresh  meats,  though  the  best  results  follow  the  use  of  0.5  percent.  0.2  per 
cent  has  germicidal  powers  when  combined  with  cold.  Sometimes  aniline 
color  is  added  to  the  sausage  meat  preservatives. 

Sodium  benzoate  is  perhaps  the  most  extensively  employed  preservative 
and  at  the  same  time  the  least  harmful,  o.i  per  cent,  added  to  food  articles, 
as  meats,  fruits,  catsups,  vinegar,  cider,  etc.,  checks  decomposition.  Gen- 
erally, however,  more  than  o.i  per  cent,  is  added,  from  0.2  to  0.5  per  cent. 


DISINFECTANTS  AND   DISINFECTION.  185 

The  percentage  of  benzoate  preservative  is  likely  to  vary  because  of  its 
volatile  nature;  canners  quite  generally  add  an  excess  knowing  that  much 
of  it  will  be  carried  off  with  the  vapors  escaping  during  the  heating  process. 
As  a  result  it  follows  that  products  declared  to  contain  o.i  per  cent,  of  benzoate 
may  upon  chemical  examination  show  the  actual  amounts  to  range  from  a 
mere  trace  (0.05  per  cent,  to  0.5  per  cent.). 

Next  to  benzoate,  salicylic  acid  is  perhaps  the  most  common  food  pre- 
servative, used  much  like  benzoate,  in  strengths  varying  from  o.io  to  2.5 
per  cent.  It  is  frequently  added  to  beers,  cordials,  wines  and  foods  (4  to  8 
grains  to  the  pint)  containing  sugars.  It  is  also  used  as  a  surgical  dressing, 
but  other  less  irritating  wound  disinfectants  are  given  the  preference. 

Crude  pyroligneous  acid  is  used  as  a  meat  preservative.  This  acid  is 
obtained  by  the  destructive  distillation  of  wood  and  contains  creosote  and 
other  tarry  matter  and  imparts  the  odor  and  taste  of  smoked  products. 
Meats,  fish,  etc.,  are  immersed  in  a  solution  of  this  acid,  dried  and  sold  as 
smoked.  This  constitutes  the  "quick"  or  "dip"  method  of  smoking  meats 
as  compared  with  the  usual  slower  method  of  exposing  the  meats  to  the 
smoke  of  slowly  burning  wood. 

The  following  are  a  few  of  the  less  commonly  employed  preservatives: 
Fluorine  compounds  are  used  in  strengths  of  from  0.03  to  0.02  per  cent. 
Alum  is  sometimes  used  in  pickling  vegetables  and  meats  (brine)  because 
of  the  hardening  effects  produced.  Copper  sulphate  is  much  used  in 
pickling  cucumbers,  peas,  string  beans  and  other  green  vegetables  for  the 
purpose  of  deepening  the  green  color.  Sodium  and  calcium  carbonate  are 
sometimes  added  to  cider  and  wine  to  check  the  souring  process  (by  com- 
bining with  the  fruit  acids).  Formic  acid  is  a  powerful  preservative.  0.014 
to  0.08  per  cent,  retards  fermentation.  Saccharin,  sucrol  and  dulcin  are 
sweetening  as  well  as  preserving  agents.  Peroxide  of  hydrogen  is  used  as  a 
preservative.  It  is  also  a  deodorant.  The  use  of  saccharin  in  food  is  no 
longer  permissible  in  the  United  States. 

6.  Insecticides  and  Other  Pest  Exterminators. 

The  farmer,  fruitgrower  and  florist  have  many  enemies  belonging  to  the 
insecta  and  to  other  divisions  of  the  animal  kingdom,  which  interfere  with 
the  productiveness  of  crops.  The  remedies  employed  against  these  pests 
are  numerous.  We  shall  mention  only  a  few  of  the  more  useful  ones,  ex- 
plaining their  action  very  briefly.  They  may  be  grouped  into  powders, 
gases,  sprays  and  washes. 

A.  Powders. — These  may  be  applied  by  the  "pepper  box"  method,  the 
material  being  placed  in  a  box,  usually  of  tin,  with  perforations,  through 
which  the  powder  sifts  on  shaking.  Or  a  blowing  device  may  be  used, 


1 86  PHARMACEUTICAL    BACTERIOLOGY. 

like  the  ordinary  bellows  box  for  blowing  insect  powder,  or  modifications 
of  this  simple  device.  A  third  method  known  as  the  sifting  method  is  much 
in  vogue  in  the  cotton  fields.  The  powder  is  placed  in  a  porous  bag  or  cloth, 
fastened  to  a  stick  and  shaken  over  the  plants  to  be  treated.  Only  three 
powders  are  used  to  any  considerable  extent,  as  follows: 

a.  Slaked  Lime. — Dry  air  slaked  lime  is  reduced  to  a  uniformly  fine 
powder  which  is  then  ready  for  use.     It  is  very  efficacious  with  all  slimy 
animals,  as  slugs  and  snails.     It  is  applied  to  plants  when  the  pests  are 
active,  that  is,  in  the  early  morning  or  in  the  evening.     Lime  is  used  where 
paris  green  is  not  permissible,  as  with  fruit  plants  and  edible  herbs. 

b.  Sulphur. — The  flower  of  sulphur  or  ground  sulphur  is  a  very  widely 
used  remedy  for  fungous  pests,  as  mildew;  also  for  the  red  spider  and  thrips. 
Sulphur  is  active  only  in  the  sunlight,  particularly  on  a  hot  day. 

The  flower  of  sulphur  gives  better  results  than  the  ground  sulphur  be- 
cause it  " sticks"  better.  It  should  be  applied  evenly  and  not  too  thickly. 
Remember  that  sulphur  dioxide  is  very  injurious  to  plants,  therefore  fumiga- 
tion by  burning  sulphur  is  out  of  the  question. 

c.  Paris  Green  and  Other  Arsenicals. — These  are  generally  not  used  in 
the  dry  powdered  form.     When  so  used  they  are  diluted  with  flour,  dust 
or  other  inert  powdered  material.     Must  be  sparingly  applied  and  evenly 
distributed,  otherwise  serious  damage  may  be  done  to  the  foliage. 

B.  Gases. — Gases  diffuse  with  great  rapidity  and  when  applied  within 
an  enclosed  space  will,  in  a  short  time,  be  uniformly  distributed  throughout 
the  enclosed  space.  The  rapid  diffusion  of  gases  is  a  great  hindrance  to 
their  practical  utilization  in  the  open  as  in  orchards,  fields  and  gardens. 
Their  use  is  quite  limited. 

a.  Carbon  Bisulphide. — This  is  not  used  with  growing  plants  though  it  is 
applied  to  stored  seeds,  and  dry  plants  and  grains,  for  the  purpose  of  killing 
insects  and  other  destructive  animals.  It  is  also  used  to  kill  pests  which 
live  in  the  soil,  as  the  grape  Phylloxera.  For  this  purpose  a  machine  is 
used  which  injects  the  bisulphide  into  the  soil.  To  destroy  pests  in  drug 
plants,  seeds  and  grain,  enclose  them  in  a  space,  place  a  dish  containing 
the  bisulphide  on  top  of  the  material.  The  vapor  being  heavier  than  the  air, 
gravitates  downward  and  soon  fills  the  entire  enclosed  area.  The  amount 
necessary  to  do  the  work  will  depend  upon  the  nature  of  the  material  to  be 
treated  and  the  tightness  of  the  enclosure.  Roughly  estimated  a  dram  of 
the  carbon  bisulphide  to  five  pounds  of  the  material  is  sufficient.  Grain- 
men  usually  apply  one  pound  to  the  ton  of  grain,  if  the  bin  is  tight. 

Carbon  bisulphide  is  one  of  the  most  effective  remedies  against  the  gopher 
and  the  ground  squirrel.  Use  the  remedy  after  a  rain  as  the  soil  is  then  less 
porous.  Pour  an  ounce  over  a  rag  or  other  porous  substance  (horse  drop- 
pings are  much  used),  stuff  this  into  the  hole  and  plug  with  a  ball  of  dirt. 


DISINFECTANTS  AND   DISINFECTION.  187 

The  bisulphide  is  also  used  to  kill  the  yellow- jacket,  which  is  very  injurious 
to  fruit,  also  the  root  crown  borer  of  the  peach,  and  to  disinfect  grapevine 
cuttings,  etc.,  etc. 

b.  Hydrocyanic  Acid  Gas. — This  is  about  the  only  gas  which  is  powerful 
enough  to  kill  insects  and  yet  not  injure  the  foliage.  It  is  used  by  covering 
the  tree,  shrub  or  bush  with  a  tent  cloth  or  canvas  which  should  be  oiled  to 
keep  in  the  vapor.  The  vessel  containing  the  chemicals  is  placed  underneath 
Exposure  of  from  thirty  to  fifty  minutes  is  usually  sufficient.  About  one 
ounce  of  potassium  cyanide  to  150  cubic  feet  of  space  is  required. 

The  gas  is  extremely  poisonous  and  is  often  destructive  to  foliage.  It  is 
preferably  applied  at  night  as  it  is  then  less  injurious  to  the  foliage. 

C.  Sprays  and  Washes. — Plant  pests  are  most  generally  destroyed  by 
spraying  agents  or  washes.  A  wash  is  really  a  more  liberal  application  of 
the  spray,  the  two  being  alike  as  to  the  results  to  be  attained  from  their  use 

For  low  plants  the  remedy  can  be  applied  by  means  of  a  sprinkling  can 
but  the  better  method  is  to  use  some  form  of  force  pump  with  spray  nozzle. 
A  good  spray  pump  should  maintain  a  uniformly  constant  as  well  as  adequate 
pressure,  should  be  simple  of  construction,  with  all  parts  readily  replaceable. 
The  nozzle  should  break  up  the  stream  into  a  fine  mist. 

It  is,  of  course,  desirable  to  get  as  much  as  possible  of  the  spray  to  remain 
on  leaf  or  stem  and  to  have  it  evenly  distributed.  If  put  on  too  abundantly 
the  fine  droplets  or  gobules  on  the  leaf  will  run. together  and  roll  off  to  the 
ground.  The  nozzle  must  not  be  held  near  the  plant  to  be  sprayed  in  order 
to  get  the  disirable  dew-like  deposit  on  the  leaf. 

For  scale  insects  a  thorough  moistening  is  necessary,  wetting  the  bark, 
the  scale  and  eggs.  In  order  to  accomplish  this  the  nozzle  must  be  held 
close. 

The  following  table  by  Woodworth  will  indicate  the  method  of  preparing 
and  using  the  more  important  spraying  solutions: 

The  well  known  Bordeaux  mixture,  so  extensively  used  as  a  spray  and 
wash  is  prepared  as  follows: 

Water,  50  gal. 

Copper  sulphate,  6  Ib. 

Unslaked  lime,  4  Ib. 

The  adhesive  properties  can  be  increased  by  adding  soft  soap  in  quantity 
equal  to  that  of  the  copper  sulphate.  It  is  also  advisable  to  dilute  the  mixture 
for  spring  spraying.  It  is  the  most  effective  and  perhaps  the  cheapest 
fungicide  that  can  be  used. 

Aphides  (plant  lice)  and  similar  plant  parasites  may  also  be  destroyed 
with  weak  solutions  of  alum  (1.5  to  2  per  cent.).  Beetles  may  be  killed  by 
sprinkling  a  mixture  of  equal  parts  of  red  lead,  sugar  and  flour,  near  their 


1 88 


PHARMACEUTICAL   BACTERIOLOGY. 


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DISINFECTANTS  AND   DISINFECTION.  189 

hiding  places,  or  a  mixture  of  borax  20  parts  and  precipitated  carbonate 
of  baryta  (native  witherite  will  not  answer  the  purpose).  A  great  variety 
of  substances  are  recommended  for  the  extermination  of  ants,  as  borax, 
camphor,  balsam  of  Peru,  spraying  with  benzine,  etc.  In  lawns  and  in  the 
open  generally  (in  ant  hills)  they  are  most  quickly  destroyed  by  means  of 
carbon  bisulphide.  This  kills  the  ants  as  well  as  the  larvae. 

The  exterminators  for  pests  of  all  sorts  is  legion  and  those  especially 
interested  must  consult  some  standard  work  on  formulas  such,  as  the  Scien- 
tific American  Cylcopedia  of  Formulas  (Hopkins) . 


CHAPTER  XII. 
STERILIZATION  AND  DISINFECTION  IN  THE  PHARMACY. 

It  is  only  within  very  recent  years  that  sterilization  in  the  pharmacy 
has  received  any  serious  attention.  Certain  pharmacopeias,  notably  those 
of  Austria  and  Beligum,  give  specific  directions  regarding  the  sterilization 
of  certain  medicamenta,  particularly  those  intended  for  hypodermic  use. 
The  German,  English,  Italian,  Swiss  and  other  pharmacopeias  give  direc- 
tions regarding  certain  sterilizing  processes  which  may  be  applied  to  a  few 
articles.  Fischer,  Stich,  Deniges,  Mario,  Schoofs  and  other  European 
investigators  have  given  the  subject  much  attention  and  have  perfected 
many  of  the  details  of  procedure. 

Some  of  the  non-official  methods  of  sterilization  are  of  very  doubtful 
practicability.  Particularly  the  methods  recommended  for  the  sterilization 
of  pharmaceutical  solutions  by  means  of  the  ultra-violet  rays  and  by  means 
of  chemical  disinfectants.  Lesure  sums  up  the  use  of  the  ultra-violet  rays 
as  follows:  "A  series  of  experiments  shows  that,  at  present,  the  ultra-violet 
rays  can  scarcely  be  regarded  as  a  practical  means  of  sterilizing  pharmaceu- 
tical solutions,  such  as  hypodermic  injections.  It  is  not  yet  possible  to 
sterilize  liquids  in  small  closed  glass  vessels,  since  the  glass  absorbs  the  rays 
of  shortest  wave  length,  which  are  precisely  those  of  most  active  sterilizing 
power.  Possibly  on  a  large  scale  solutions  could  be  sterilized  in  bulk  and 
then  filled,  in  vacuo,  into  sterilized  small  receivers.  The  rays  might  be 
useful  for  substances  which  are  decomposed  by  treatment  in  the  autoclave. 
Some  substances  are,  however,  so  readily  decomposed  by  ultra-violet  rays, 
that  their  solutions  can  never  be  sterilized  therewith.  Such  are  solutions 
of  quinine  salts,  of  mercuric  iodide,  of  atoxyl,  of  eserine,  of  apomorphine 
and  some  glucosides,  as  for  example  gentiopicrin.  Opaque  solutions  and 
suspensions  of  solids  cannot  be  thus  sterilized.  The  permeability  of  the 
different  solutions  to  the  rays  also  varies  very  greatly.  Apart  from  the 
question  of  decomposition,  it  is  found  that,  in  the  case  of  gentiopicrin, 
completely  sterile  solutions  were  not  obtained  even  after  an  exposure  of 
half  an  hour;  on  the  other  hand  ancubin  solutions  were  completely  sterilized 
in  thirty  seconds."  The  decomposition  changes  due  to  the  ultra-violet  rays 
are  not  clearly  understood.  The  indications  are  that  there  are  no  very 
marked  chemical  changes  in  such  substances  as  cocaine  and  pilocarpin 
hydrochloride  after  three  hours'  exposure.  Arbutin  shows  a  change  in  a  few 

190 


STERILIZATION  AND   DISINFECTION   IN   THE   PHARMACY.  IQI 

minutes.  There  is  so  much  uncertainty  as  to  the  results  that  the  method 
cannot  as  yet  be  recommended  for  practical  use. 

The  addition  of  disinfectants  to  medicines  for  purposes  of  sterilization 
has  recently  received  some  attention.  The  use  of  formaldehyde,  ether, 
chloroform  and  alcohol,  have  been  recommended,  each  having  its  special 
use  in  practice.  The  general  criticisms  made  regarding  the  use  of  the  ultra- 
violet rays  also  apply  here.  Currie  recommends  a  formalin  method  as 
follows:  applicable  to  infusions  of  calumba,  gentian,  quassia  and  senega 
"The  infusions  of  calumba  and  quassia  are  simply  evaporated  to  one-eighth 
of  their  bulk,  filtered,  and  4  minims  of  the  ordinary  40  per  cent,  solution  of 
formaldehyde  added  to  each  fluid  ounce  of  the  concentrated  infusion.  On 
dispensing,  the  requisite  amount  is  put  in  a  shallow  basin  and  brought 
sharply  to  the  boil,  thus  dissipating  the  formaldehyde.  The  infusion  is  then 
diluted  to  the  normal  strength  with  sterilized  distilled  water.  Infusion  of 
gentian  is  made  from  gentian  root  alone,  and  concentrated.  To  this  is 
added  essence  of  lemon  (i  in  10),  and  the  official  tincture  of  orange  in  the 
proportion  of  2  fluid  drams  of  the  former  and  i  fluid  ounce  of  the  latter  to 
each  pint  of  the  infusion.  There  is  also  added  4  minims  of  40  per  cent,  solu- 
tion of  formaldehyde  to  each  fluid  ounce  of  infusion.  Infusion  of  senega 
is  concentrated  by  evaporation  and  to  prevent  precipitation,  5  grains  of 
potassium  bicarbonate  are  added  to  each  fluid  ounce  of  the  concentrated 
solution,  and  4  minims  of  40  per  cent,  solution  of  formaldehyde.  In  case 
of  both  gentian  and  senega  infusion,  the  formaldehyde  is  dissipated  at  the 
time  of  dispensing,  in  the  manner  already  described.  The  advantages  of 
this  process  are  ease  of  manipulation,  cheapness,  and  the  certainty  of  the 
antiseptic  condition  of  the  infusion  while  being  kept  in  stock  and  until  dis- 
pensed. The  quantity  of  formaldehyde  remaining  in  the  diluted  infusion 
is  infinitesimal,  and  may  be  ignored  for  all  practical  purposes." 

It  is  known  that  weak  solutions  of  hypodermic  and  intravenous  solutions, 
unless  sterilized,  will  show  numerous  bacteria  upon  standing  for  a  time. 
One  per  cent,  solutions  of  pilocarpin,  atropin,  cocaine,  morphine,  and  fluid- 
extract  of  ergot  have  been  found  to  contain  millions  of  bacteria  per  c.c. 
However,  loper  cent,  iodoform  glycerin,  camphorated  oil  (i  in  10),  solutions 
of  apomorphin  (0.2  in  20),  quinine  (i  in  10),  antipyrin  (5  in  10),  cocaine 
(10  per  cent.)  are  usually  quite  free  from  bacteria.  In  a  general  way  the 
bacterial  content  of  medicinal  solutions  decreases  directly  with  the  degree  of 
concentration.  Pus  microbes  die  at  once  in  ether  and  in  a  saturated  solution 
of  quinine,  whereas  they  remain  active  in  a  10  per  cent,  solution  of  cocaine. 
A  2  per  cent,  solution  of  morphine  kills  pus  microbes  in  twenty-four  hours, 
while  pure  glycerin  kills  them  only  after  an  exposure  of  six  to  eight  days. 

A  perfectly  safe  rule  for  the  pharmacist  is  to  consider  all  medicamenta 
which  he  handles  and  which  he  may  be  called  upon  to  dispense,  as  being 


IQ2  PHARMACEUTICAL   BACTERIOLOGY. 

possibly  contaminated  and  to  sterilize  and  disinfect  all  articles  which  in  his 
judgment  as  a  qualified  pharmacist  may  require  such  treatment,  in  so  far 
as  it  is  practically  possible.  The  retail  pharmacist  must  not  place  too  much 
confidence  in  the  assertions  of  comparatively  little  known  manufacturers 
and  wholesale  houses,  regarding  the  sterile  conditions  of  the  articles  which 
they  may  supply. 

The  medicines  found  in  a  drug  store  and  dispensed  by  the  pharmacist 
may  be  grouped  as  follows: 

A.  Medicines  which  do  not  Generally  Require  Sterilization. 

a.  For  internal  administration  per  mouth.     They  may  be  contaminated 
or  may  become  contaminated  on  standing  for  a  time.     Such  medicines 
should  be  rejected.     Do  not  attempt  to  render  them  usable  by  sterilization. 

b.  Mouth  washes  and  gargles. 

c.  Enemas.     Enemas  for  young  children  and  such  enemas  as  are  to  be 
applied  to  inflamed  or  otherwise  pathologic  conditions  of  the  intestinal 
mucous  membrane,  should  be  sterilized. 

d.  Medicamenta  which  are  to  be  applied  to  the  intact  skin,  or  to  the 
scalp. 

B.  Medicines  Which  Require  Sterilization. 

a.  Those   intended  for  intravenous   and   hypodermic   use.     Not   only 
must  these  be  absolutely  sterile  but  they  must  be  in  perfect  solution,  before 
using. 

b.  Those  to  be  applied  to  cuts,  bruises,  abrasions,  wounds,  ulcers,  sores, 
and  to  the  broken  skin  generally. 

c.  Those  to  be  applied  to  inflamed  mucous  membranes,  as  enemas, 
douches,  etc. 

d.  Solutions  for  the  irrigation  of  the  bladder. 

e.  Eye  medicines,  as  washes  and  other  solutions,  intended  for  direct 
application  to  the  eye. 

i.  Methods  of  Sterilization. 

The  following  methods  of  sterilization  are  applicable  in  the  pharmacy 
and  should  be  consistently  practised : 

A.  Sterilization  of  Containers. — The  glassware  and  other  containers  used 
in  the  pharmacy  should  be  cleaned  and  sterilized  as  follows: 

a.  Bottles  and  Glassware  Generally. — Wash  and  rinse  in  warm  water  to 
remove  dust,  dirt,  sand,  straw,  etc.,  then  wash  and  rinse  in  hot  water  with 
2  to  5  per  cent,  sodic  hydrate.  Neutralize  the  sodic  hydrate  by  washing 
and  rinsing  in  2  to  5  per  cent,  hydrochloric  acid.  Finally  wash  and  rinse 
in  hot  sterile  water  and  allow  to  drain.  Wipe  dry  and  plug  lightly  with 


STERILIZATION  AND   DISINFECTION   IN   THE   PHARMACY.  193 

cotton.  Place  the  plugged  bottles  in  a  hot-air  sterilizer  and  heat  for  one 
hour  at  120°  C.  to  130°  C.  Keep  these  cleaned,  sterilized,  and  cotton-plugged 
bottles  in  clean  container  in  a  dry  clean  store-room,  until  wanted  foroise. 

b.  Porcelain  and  Similar  Containers. — May  be  cleaned  and  sterilized 
like  glassware.     Plugging  with  cotton  is  as  a  rule  inadmissible. 

c.  Large  Flasks,  Jugs,  Etc. — Large   containers  are   as   a   rule   difficult 
to  sterilize  and  for  this  very  reason  are  often  subject  to  special  neglect. 
Proceed  much  as  for  bottles,  observing  greater  caution  as  to  changes  in 
temperature.     Large    bottles,   carboys  and  similar  containers    cannot  be 
sterilized  by  means  of  boiling  hot  water  as  they  are  very  apt  to  crack.     They 
may  be  sterilized  by  means  of  carbolic  acid  (5  per  cent.),  lysol  (1.5  per  cent.) 
or  formaldehyde  (4  per  cent.),  then  thoroughly  rinsed  in  sterile  water,  allowed 
to  drain,  plugged  with  cotton,  carefully  heated  in  hot-air  sterilizer  for  one 
hour  or  more  at  115°  to  120°  C.     Cool  gradually. 

d.  Tin  Containers. — Wash  and  rinse  thoroughly  in  water;  boil  for  thirty 
minutes,  drain  and  dry  and  sterilize  in  dry-air  sterilizer  for  one  hour  at  100°  C. 

B.  Sterilization  of  Apparatus  and  Tools. — It  is  of  the  highest  importance 
that  mortar  and  pestle,  spatulas,  percolators,  pill  and  suppository  machines, 
mixing  plates,  etc.,  etc.,  should  be  clean  and  sterile.     This  means  a  liberal 
use  of  hot  water,  green  or  soft  soap,  and  clean  towels.     The  sink,  the  floor 
of  the  dispensing  room,  the  tables,  chairs,  desks,  in  fact  everything  in  and 
about  the  dispensing  room  should  be  scrupulously  clean. 

C.  Sterilization  of  Corks  and  Other  Stoppers  for  Containers. — It  would  be 
energy  wasted  to  clean  and  sterilize  the  containers  if  the  stoppers  are  not 
also  clean  and  sterile.     Sterilize  corks  by  washing  in  hot  60  to  75  per  cent, 
alcohol,  drain  and  heat  in  hot-air  sterilizer  for  one  hour  at  130°  C.     Keep 
these  corks  in  sterilized  wide-mouthed  ground-glass  capped  bottles.     Take 
out  corks  as  wanted  by  means  of  a  sterile  pair  of  pincers,  not  by  means  of 
fingers.     Other  stoppers,  as  of  glass,  of  wood,  of  rubber,  must  also  be 
cleaned  and  sterilized.     Rubber  caps,  rubber  stoppers,  and  other  rubber 
goods  may  be  sterilized  by  boiling  in  water  for  thirty  minutes. 

D.  Sterilization  of  Surgical  Supplies. — a.  Bandaging  materials,  cotton, 
absorbent  gauze,  etc.,  may  be  sterilized  by  wrapping  in  cheese  cloth  or 
filter  paper,  first  placing  a  grain  of  fuchsin  or  other  aniline  dye  in  the  center 
of  the  package  (wrapped  in  paper  or  cloth),  and  sterilizing  in  steam  for  one 
hour.     The  dye  particle  is  introduced  as  a  test  object  to  ascertain  if  the 
steam  has  penetrated  the  entire  package.     If  it  has  penetrated  the  entire 
package  it  will  be  indicated  by  a  spreading  of  the  color.     Afterward,  dry 
for  one  hour  at  100°  C.  in  the  hot-air  sterilizer.     For  this  purpose  the  form 
of  Arnold  steam  sterilizer  shown  in  Fig.  18  will  be  found  very  useful. 

b.  Sewing  materials,  such  as  needles,  forceps,  catgut,  etc.,  require  careful 
sterilization  before  using.     All  metal  instruments  and  appliances,  including 


IQ4  PHARMACEUTICAL   BACTERIOLOGY. 

silver  wire,  can  be  sterilized  in  5  per  cent,  carbolic  acid  if  necessary  or  they 
may  be  boiled  for  30  to  50  minutes.  Wipe  perfectly  dry  with  sterile  towels 
and  place  in  hot-air  sterilizer  for  one  hour  at  100°  C.  In  order  to  keep  them 
in  sterile  condition  for  immediate  use  they  must  be  kept  wrapped  in  sterilized 
cloth  or  cotton. 

c.  Catgut  requires  thorough  sterilization  as  not  infrequently  spores  of 
disease  germs  (as  anthrax)   are  present.     The  so-called  cumol   (cumene) 
method  of  catgut  sterilization  is  quite  generally  adopted  in  the  hospitals  of 
Germany  and  of  other  European  countries.     Wind  the  catgut  in  the  usual 
ring  form,  dry  in  hot-air  sterilizer  for  two  hours  at  70°  C.,  place  rings  in  a 
vessel  (beaker,  etc.)  with  cumol  on  sand-bath  and  heat  to  155°  C.  or  165°  C. 
(the  boiling-point  of  cumol),  turn  off  the  gas  and  allow  to  remain  in  the  hot 
cumol  for  one  hour.     The  cumol  dish  should  be  covered  with  a  fine  mesh 
wire  screen  to  guard  against  catching  fire.     Take  the  catgut  rings  out  of  the 
cumol  by  means  of  sterile  pincers  and  place  in  benzine  for  three  hours,  then 
allow  the  benzine  to  evaporate  in  sterile  Petri  dishes. 

d.  Silver  catgut  is  preferably  sterilized  in  i  per  cent,  silver  citrate  (itrol) 
or  i  per  cent,  silver  lactate  (actol),  allowing  it  to  remain  for  six  hours, 
which  destroys  even  the  anthrax  spores.     Next  expose  the  catgut  to  light 
(in  sterile  dishes)  for  a  day  or  two,  then  wind  or  fasten  on  glass  and  preserve 
in  95  per  cent,  alcohol  with  10  per  cent,  glycerin.     Actol  and  itrol  ionize 
silver  far  less  actively  than  silver  nitrate,  hence  their  preference. 

e.  Catheters,  drainage  tubing  and  other  rubber  materials  are  sterilized 
by  boiling  in  water  with  5  per  cent,  sodic  hydrate.     Rubber  goods  will  not 
stand  prolonged  and  frequent  boiling.     Do  not  sterilize  metal  ware  with 
rubber  goods. 

e.  Sterilization  of  Medicines. — As  a  rule,  medicines  which  are  prepared 
under  aseptic  surroundings  and  conditions  do  not  require  sterilization. 
However,  the  ideal  conditions  rarely  exist  and  subsequent  sterilizations 
become  desirable  and  even  necessary. 

Tooth  powders,  dusting  powders  and  similar  substances  may  be  ster- 
ilized at  a  dry  temperature  of  70°  C.,  for  three  to  four  hours.  Salves  and 
pastes  are  difficult  to  sterilize.  Low  temperatures  (from  60°  C.  to  70°  C.) 
for  several  hours  may  be  employed. 

Solutions  for  subcutaneous  injection,  for  wound  irrigation,  for  bladder 
irrigation,  solutions  of  boric  acid,  of  tannic  acid,  aquae,  normal  salt  solu- 
tions and  all  weaker  solutions  of  chemicals,  intended  for  washes  and  irriga- 
tion in  surgery,  should  be  sterilized  by  boiling  for  five  minutes.  Strong 
solutions  of  chemicals  (as  acids,  alkalies,  etc.)  do  not  require  sterilization 
as  they  are  themselves  strongly  germicidal. 

Alkaloidal  and  glucosidal  solutions,  and  solutions  of  alkaloidal  salts, 
tinctures  and  fluidextracts,  should  be  carefully  filtered  and  sterilized  in 


STERILIZATION  AND   DISINFECTION  IN  THE   PHARMACY.  195 

sealed  containers  at  a  temperature  of  60°  C.,  one  hour  each  day  for  six  days. 
Concentrated  alkaloidal  solutions  may  be  similarly  sterilized.  It  is  not 
advised  to  employ  a  higher  temperature  for  these  substances  inasmuch  as 
the  decomposition  changes,  if  any,  which  may  take  place  at  100°  C.  are 
not  clearly  understood.  To  be  on  the  safe  side,  the  lower  temperature 
(60°  C.)  should  be  employed. 

In  the  case  of  solutions  or  emulsions  for  hypodermic  use,  prepared  with 
oil,  the  oil  is  first  to  be  treated  with  alcohol  (95  per  cent.)  to  remove  the  oleic 
acid.  Oily  solutions  of  calomel,  yellow  oxide  of  mercury,  lecithin,  and  of 
camphor  are  to  be  prepared  with  sterile  materials,  then  placed  in  a  boiling- 
water-bath  for  ten  minutes  or  in  an  air-bath  at  100°  C.  An  interesting 
requirement  is  exacted  by  the  Italian  Pharmacopeia  as  regards  the  glass- 
of  the  containers  for  hypodermic  injections:  Ten  to  twelve  ampuls  or 
five  or  six  bottles  are  filled  with  a  clear  solution  of  i  per  cent,  mercuric 
chloride,  then  sealed.  They  are  then  left  in  an  autoclave  at  112°  C.  for 
half  an  hour,  at  the  expiration  of  which  time  no  brownish  turbidity  should 
be  perceptible. 

Some  of  the  points  pertaining  to  the  sterilization  of  alkaloidal,  glucosidal 
and  other  substances  which  are  quite  readily  decomposed  or  altered  by 
light  and  heat,  will  be  treated  under  ampuls. 

2.  Preparation  of  Ampuls. 

Ampuls  (Lat.  ampulla ;Fr.  ampoule; — a  flask)  are  small  glass  containers 
filled  with  medicinal  substances  usually  in  solution.  These  have  come  into 
great  prominence  within  recent  years,  due  to  the  methods  of  sterilization 
now  required  and  practised  in  well  regulated  pharmacies.  Ampuls  are 
really  nothing  more  than  very  small  flasks,  the  size  being  suited  to  single 
doses  of  the  medicine,  as  a  rule.  They  were  introduced  into  France  about 
thirty  years  ago  by  Limousin  and  have  now  come  into  general  use  in  France, 
Italy,  Spain,  Holland  and  England.  It  is  only  recently  that  they  have 
come  into  use  in  the  United  States.  C.  A.  Mayo  was  among  the  first  Amer- 
ican writers  to  publish  the  first  more  complete  information  regarding  their 
origin,  manufacture  and  use.  (See  Proc.  A.  Ph.  A.,  vol.  57,  1909.)  They 
are  generally  adopted  by  the  navies  and  armies  of  all  civilized  countries, 
because  of  the  advantage  which  they  offer  for  the  preservation,  storage  and 
transportation  of  all  manner  of  medicines,  particularly  those  which  require 
sterilization  and  which  are  generally  wanted  for  immediate  administration. 
From  the  standpoint  of  the  physician  they  are  wonderfully  convenient  and 
are  great  time  savers. 

Ampuls  may  have  any  desired  capacity,  from  i  c.c.  up  to  100  c.c.,  and 
more,  but  the  more  usual  capacities  are  i  c.c.,  2  c.c.,  5  c.c.,  and  10  c.c.  They 
are  made  of  alkali-free  glass,  white  or  colored  (amber) .  Those  supplied  by 


ig6 


PHARMACEUTICAL   BACTERIOLOGY. 


French,  German  and  Italian  makers  are  of  different  forms,  as  flask-like, 

bulb-like,  spindling,  globose,  etc. 

The  following  are  some  of  the  reasons  why  ampuls  have  come  into  use: 
a.  Most  of  the  liquid  medicamenta  and  those  which  are  to  be  dissolved 

before  using  have  little  or  no  antiseptic  power  and  under  the  usual  conditions 


0 

FIG.  75. — Making  ampuls,  a,  Piece  of  glass  rod  to  make  two  ampuls;  b,  rod  a  heated  in  the 
middle  and  nearly  drawn  apart;  c,  d,  two  half  ampuls  filled;  e,f,  the  completed  ampuls. 

readily  become  highly  contaminated  with  different  organisms.     The  use 
of  such  contaminated  medicines  has  led  to  serious  infections. 

b.  The  necessity  of  direct  administration  of  medicinal  solutions,  by  hypo- 
dermic, intramuscular  and  intravenous  injection,  is  due  to  the  desirability 
of  getting  prompt  therapeutic  effects. 

c.  The  direct  (hypodermic,  intramuscular  and   intravenous)    adminis- 


STERILIZATION  AND    DISINFECTION   IN    THE    PHARMACY.  197 

tration  of  medicamenta  is  very  frequently  necessary  because  administration 
per  mouth  is  impossible  or  undesirable. 

As  a  rule  the  pharmacist  will  purchase  ampuls,  ready  for  immediate  use 
by  the  physician,  from  some  reliable  wholesale  manufacturing  house.  In 
certain  districts  and  under  certain  conditions  this  may  not  always  be  possible, 
in  which  case  the  pharmacist  must  prepare  the  ampuls.  The  pharmacist 
should  be  prepared  to  make  all  ampuls  which  may  be  desired  by  the  physi- 
cians in  his  community.  The  following  suggestions  can  be  carried  out 
readily: 

A.  Glass  Tubing. — Ampuls  can  readily  be  made  from  ordinary  alkali- 
free  glass  tubing,  selecting  rods  of  a  diameter  to  make  ampuls  of  i  c.c.,  2  c.c., 
5  c.c.,  and  10  c.c.  capacity.     This  tubing  can  be  secured  from  any  chemical 
or  pharmaceutical  supply  house.     Select  rods  which  are  quite  free  from 
bubbles  and  of  fairly  uniform  diameter  and  thickness. 

B.  Breaking  the   Tubing  into  Suitable  Lengths. — Break  the   tubing  in 
lengths  of  from  five  to  six  inches,  by  filing  a  scratch  with  a  small  file  and 
breaking,  with  the  hands  protected  by  gloves  to  avoid  injury  by  small  bits 
of  glass. 

C.  Sterilizing  and  Neutralizing  the  Glass  Tubing. — Place  the  lengths  of 
glass  rods  into  water  with  5  per  cent,  of  soda  and  boil  for  thirty  minutes. 
Neutralize  in   5   per  cent,  hydrochloric  acid,  rinse  thoroughly  and  again 
boil  in  distilled  water.     Let  drain  until  dry.     May  be  dried  in  hot-air  ster- 
ilizer at  140°  C. 

D.  Making  the  Half  Ampul. — Take  one  glass  tube  and  heat  the  middle 
part  in  a  bunsen  burner  with  rotation  until  red  hot  and  soft,  and  pull  apart 
with  a  fairly  quick  strong  pull.     Break  off  the  thin  hairlike  ends  and  hold 
the  tips  in  the  flame  to  seal  them  securely.     A  smalt  bead  should  form  as 
shown  in  Fig.  75,  c,  d,  e,  f.     A  little  practice  with  a  steady  hand  is  neces- 
sary to  do  this  neatly.     The  half  ampuls  (one  end  open,  the  other  sealed  as 
explained)  are  now  laid  aside  in  a  sterile  box  or  other  container,  until  ready 
to  be  filled.     Or  the  two  ends  of  the  ampul  can  be  reduced  to  a  capillary 
tube  as  follows.     Heat  the  glass  tubing  in  the  blow-pipe  flame,  beginning 
at  one  end,  until  soft  and  draw  out  a  short  distance  with  a  firm  pull.     Heat 
at  a  point  about  i  to  3  inches  from  the  narrowing  portion  of  the  glass  tube 
and  repeat  as  before.     Repeat   this  until  there    are  a  series  of  tubes  of 
normal  diameter  with  capillary  connections.     Breaking  these  apart  with 
the  aid  of  a  file,  gives  empty  ampuls  open  at  the  two  capillary  ends. 

E.  Filling  the  Half  Ampuls. — This  can  be  done  by  means  of  a  burette,  a 
pipette  or  a  medicine  dropper.     The  burette  has  many  advantages.     Many 
ampuls  can  be  filled  from  one  burette,  the  exact  amounts  can  easily  be 
measured.     The  pipette  is  far  less  convenient  than-  the  burette  and  is  more 
easily    contaminated.     A  well    graduated   medicine    dropper  is  very  con- 


198  PHARMACEUTICAL    BACTERIOLOGY. 

venient,  but  all  things  considered  the  burette  is  recommended.     The  points 
to  be  kept  in  mind  are. 

a.  The  finished  ampul  should  not  be  more  than  three-quarters  full. 
The  length  (of  untapered  portion  of  tube)  of  a  neat  looking  ampul  is  about 
three  or  four  times  the  diameter  of  the  tubing  used. 

b.  In  filling,  introduce  at  least  10  per  cent,  more  than  the  actual  dose 
required,  that  is,  the  i  c.c.  tube  should  contain  i.io  c.c.;  the  5  c.c.  tube  should 
contain  5.50  c.c.  of  the  medicinal  substance,  etc.     This  is  to  make  sure  that 
the  physician  may  get  a  full  i  c.c.,  5  c.c.,  etc.,  dose  after  allowing  for  unavoid- 
able loss  (portion  clinging  to  inside  of  ampul,  remaining  in  narrowed  ends, 
etc.). 

c.  In  filling  do  not  allow  any  of  the  liquid  to  come  in  contact  with  the 
upper  end  (open  end)  of  the  tube  as  that  would  interfere  with  sealing. 

There  are  many  different  methods  for  filling  ampuls  which  may  be 
classed  under  three  heads;  filling  by  gravity,  by  pressure,  and  by  vacuum; 
the  latter  two  being  but  modifications  of  the  same  principle  involved.  There 
are  on  the  market  (France,  Holland,  Germany)  several  devices  made  ex- 
pressly for  filling  and  sealing  ampuls. 

F.  Sealing  the  Filled  Half  Ampuls. — This  is  done  by  means  of  suitable 
side-flame  blow-pipe  burner,  pinching  together  and  drawing  out  the  soft  end 
of  the  glass  by  means  of  pincers  and  sealing  in  same  manner  as  the  other  end. 
Do  not  upend  the  ampul  until  it  is  cool,  to  avoid  cracking  the  glass. 

G.  Sterilizing  the  Ampuls. — The  hypodermic  and  other  solutions  usually 
put  up  in  ampuls  can  be  divided  into  three  classes  or  groups  according  to 
the  degree  of  heat  which  may  or  must  be  used  in  sterilizing,  namely,  those 
which  cannot  withstand  a  temperature  above  60°  C.,  those  which  can  be 
sterilized  at  100°  C.,  and  those  which  may  be  sterilized  in  an  autoclave  at 
120°  C.     Inasmuch  as  the  autoclave  is  rarely  usable  and  also  because  the 
ordinary  steam  temperature  (100°  C.)  will  meet  all  of  the  requirements  of 
the  autoclave,  the  latter  piece  of  apparatus  may  be  left  out  of  consideration 
by  the  practising  pharmacist. 

To  bring  about  a  complete  sterilization  of  the  ampuls,  the  discontinued 
or  fractional  method  should  in  all  cases  be  carried  out.  Place  the  ampuls 
in  a  container  (beaker,  tumbler,  etc.)  with  water  to  which  enough  methyl 
blue  or  fuchsin  has  been  added  to  give  it  a  very  marked  color  and  sterilize 
as  follows:  If  a  temperature  of  60°  C.  is  to  be  used,  apply  this  temperature 
(in  incubator  with  Reichert  thermo  regulator)  for  one  hour  each  day  for 
four  to  eight  days.  Some  manufacturers  recommend  a  period  of  ten  days. 
If  the  100°  C.  is  to  be  used,  apply  this  temperature  (in  an  ordinary  Arnold 
steam  sterilizer)  for  from  20  to  30  minutes  once  each  day  for  three  days. 
Should  the  autoclave  be  used,  an  exposure  for  a  period  of  20  minutes  at 
120°  C.  is  sufficient  to  kill  all  organisms,  including  spores. 


STERILIZATION    AND    DISINFECTION    IN    THE    PHARMACY.          199 

It  is  of  vital  importance  in  preparing  liquids  for  hypodermic  and  intra- 
venous injection  to  have  absolutely  perfect  solutions.  There  must  be  no 
insoluble  particles  as  these  might  cause  serious  harm.  After  the  solutions 
are  made  they  should  be  forced  through  a  Berkefeld  or  Pasteur- Chamber- 
land  filter.  All  operations  should  be  done  under  aseptic  conditions,  using 
only  chemically  pure  materials  and  boiled  distilled  water.  If  the  contents 
of  the  ampuls  become  cloudy  after  sterilization  or  if  the  inside  of  the  glass 
tubes  show  opacities  something  is  wrong  and  such  ampuls  should  be  rejected. 
Also  reject  all  " leaks,"  indicated  by  the  aniline  color  which  will  appear  on 
the  inside  of  the  tube. 

The  finished  ampuls  are  now  ready  for  use.  The  physician  simply 
breaks  off  one  end  of  the  ampul,  inserts  the  hypodermic  needle  (sterilized), 
upends  the  ampul  and  aspirates  the  contents  of  the  ampul  into  the  syringe 
by  simply  drawing  down  the  piston.  A  second  method  is  to  remove  the 
piston  from  the  syringe  tube,  break  off  one  end  of  the  ampul,  insert  this  end 
into  the  open  end  of  the  piston  tube,  break  off  the  other  end  of  the  ampul, 
whereupon  the  contents  will  flow  into  the  piston  tube;  afterward  replace 
the  piston  rod.  In  this  latter  method  great  care  must  be  observed  so  as  not 
to  get  small  particles  of  broken  glass  into  the  hypodermic  syringe. 

Use  white  glass  for  making  ampuls.  Those  filled  with  solutions  which 
are  affected  by  light  may  be  kept  in  an  amber-colored  bottle  or  other  con- 
tainer which  is  impervious  to  light. 

The  following  substances  are  commonly  put  up  in  ampuls.  Many 
others  can  be  so  put  up.  Each  ampul  should  contain  enough  material  for 
one  dose  or  for  one  application,  as  the  case  may  be.  In  the  columns  to  the 
right  are  given  the  sterilization  temperatures;  the  preferred  or  only  usable 
temperatures  being  given  in  degrees,  the  permissible  method  being  indi- 
cated by  "Yes"  and  the  inadmissible  method  being  indicated  by  "No." 
In  case  of  doubt  it  is  always  advisable  to  use  the  lower  temperature  (60°  C., 
hourly  for  from  four  to  eight  days) . 

Sterilizing  Temperatures 

Name  of  Article 

Incubator  Steam  Steam  (auto- 

60°  C.  100°  C.         clave)  120°  C. 

No 
No 
No 
No 
No 
No 
No 
No 


Adrenalin  

Yes 

100°  C. 

Alkaloidal  salts  generally  
Alkaloids  generally 

60°  C. 

60°  C. 

Yes? 

Yes? 

Antitoxins  

60°  C. 

No 

Argyrol  

60°  C. 

Yes? 

Arsacetin. 

Yes 

100°  C. 

Arsenate  of  iron 

Yes 

100°  C. 

Arsenic  

Yes 

100°  C. 

2OO 


PHARMACEUTICAL   BACTERIOLOGY. 


Name  of  Article 

Sterilizing   Temperatures 

Incubator 
60°  C. 

Steam 
100°  C. 

Steam  (auto- 
clave) I20°C. 

Atoxyl  

60°  C. 
60°  C. 
60°  C. 
60°  C. 
Yes 
60°  C. 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
60°  C. 
60°  C. 
60°  C. 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
Yes 
60°  C. 
60°  C. 
Yes 
Yes 
Yes 
60°  C. 
60°  C. 
60°  C. 
60°  C. 

No 
Yes? 
No 
No 
100°  C. 
No 
100°  C. 
100°  C. 
100°  C. 
No 
No 
No 
No 
No 
100°  C. 
No 
No 
100°  C. 
100°  C. 
No 
No 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
100°  C. 
No 
No 
100°  C. 
No 
No 
100°  C. 
100°  C. 
100°  C. 
No 
No 
No 
No 

No 
No 
No 
No 
No 
No 
No 
Yes 
Yes? 
No 
No 
No 
No 
No 
No 
No 
No 
No? 
No 
No 
No 
No 
No 
Yes 
Yes 
No 
No 
No 
Yes 
Yes 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 
No 

Atropin  .... 

Bacterins  

Cacodylates  

Caffeine  . 

Caffeine  benzoate 

Calomel  cream  

Camphorated  oil  

Chemicals  in  solution 

Cocaine 

Duboisine 

Ergot        .            .  .      .  .          

Eserine  sulphate 

Eucaine  

Gelatin 

Glucosides 

Glycerophosphates          

Grey  oil 

Gums 

Hyoscine     

Iron  cacodylate 

Mercury  benzoate  

Mercury  cacodylate  

Mercury  salicylate 

Mercury  sozo-iodolate  

Mercurial  salts  generally.  ...         

Morphine  

Mucilaginous  substances 

Normal  salt  solution  

Oils 

Paraffins  

Quinine  .... 

Physostigmine 

Salvarsan  

Scopalamine  

Sera  

Sodium  cacodylate  

Stovaine 

Strophantin  

Strychnine  

Toxins         ... 

Tryosin 

Vaccines.  . 

STERILIZATION   AND    DISINFECTION    IN    THE    PHARMACY.  2OI 

Empty  ampuls  of  German  and  French  make  can  be  secured  from  dealers 
in  glassware  and  chemical  supplies,  likewise  the  appliances  for  filling  and 
sealing.  These  ready-made  empty  and  filled,  ampuls  vary  in  form  as 
already  indicated.  Those  with  a  flat  bottom  and  which  will  remain  stand- 
ing when  placed  on  a  flat  surface  are  preferred  by  some  physicians. 

The  ready-made  empty  ampuls  (still  sealed)  may  be  sterilized  by  boiling 
for  fifteen  minutes  in  a  5  per  cent,  solution  of  phenol,  rinsing  thoroughly 
in  boiling  hot  sterile  water,  draining  and  drying.  With  the  aid  of  a  small 
sharp  file,  break  off  the  tips  of  the  ampuls  to  be  filled.  Place  them  in  dis- 
tilled water,  bring  to  a  boil,  take  vessel  from  the  fire  for  a  few  moments, 
pour  cold  distilled  water  upon  the  empty  floating  ampuls,  a  partial  vacuum 
is  produced  in  the  interior  of  the  ampuls  and  they  quickly  fill  with  water. 
Now  boil  for  thirty  minutes.  When  water  is  sufficiently  cool  take  out  the 
ampuls,  shake  out  the  water  and  dry  in  the  hot-air  sterilizer  at  100°  C.  They 
are  then  ready  to  be  filled,  sealed  and  finally  sterilized  in  the  manner  already 
described.  An  ordinary  sterilized  hypodermic  syringe  will  be  found  very 
satisfactory  for  filling  the  ampuls.  The  suggestions  regarding  the  amount 
of  material  to  be  placed  in  the  ampul,  sealing,  sterilization,  use  of  the  aniline 
solution,  etc.,  already  given,  also  apply  here. 


CHAPTER  XIII. 

COMMUNICABLE  DISEASES  WITH  SUGGESTIONS  ON   PRE- 
VENTIVE MEDICINE. 

The  pharmacist  should  be  prepared  to  assist  the  physician  and  the 
health  authorities  in  the  enforcement  of  the  sanitary  rules  and  regulations. 
To  this  end  he  should  be  informed  as  regards  the  source  of  the  more  im- 
portant contagious  and  infectious  diseases  and  the  causes  of  epidemics  and 
the  means  available  to  prevent  or  to  combat  such  conditions.  This  does  not 
mean  that  the  pharmacist  must  have  a  full  knowledge  of  the  pathology 


FIG.  76. — Bacillus  botulinus.  This  bacillus  causes  botulism,  a  form  of  meat  poisoning. 
There  are  numerous  cases  of  poisoning  resulting  from  eating  infected  meats.  It  should  be 
kept  in  mind,  however,  that  meat  may  not  be  decomposed  and  may  be  without  bacilli  and 
yet  ptomaines  may  be  present.  Therefore  absence  of  bacilli  and  of  bad  odor  does  not  prove 
that  the  meat  is  wholesome.  Meat  from  animals  recently  killed,  which  has  been  well 
cared  for  and  which  is  without  bad  odor  and  shows  no  bacilli,  is  in  all  probability  whole- 
some. Ham,  canned  meats,  cold  storage  meats,  etc.,  may  have  taken  up  toxins  from 
contaminated  meats,  thus  being  made  unfit  for  consumption  even  though  no  bacteria  are 
found. 

and  therapeutics  of  disease.  He  should  have  at  least  a  general  knowledge 
of  the  causes  of  disease  in  order  that  he  may  assist  in  applying  the  means 
for  preventing  disease.  It  is  not  within  the  province  of  the  pharmacist  to 
cure  disease,  but  he  should  be  a  potent  factor  in  preventive  medicine. 

A  contagious  disease  is  one  which  is  readily  communicable,  from  one 
person  or  animal  to  another,  either  through  direct  contact  or  very  close 
proximity.  An  infectious  disease  is  communicable  through  a  considerable 

202 


COMMUNICABLE  DISEASES.  203 

interval  of  space.  Itch,  for  example,  is  contagious,  but  not  in  the  least 
infectious,  whereas  whooping-cough  is  infectious,  but  not  contagious. 
Some  diseases  are  both  contagious  and  infectious,  as  small-pox  and  diph- 
theria. Malaria  and  yellow  fever  are  infectious,  but  not  in  the  least  con- 
tagious. However,  the  distinctions  between  infectious  and  contagious  are 
often  not  very  clear.  It  would  be  better  to  discontinue  these  terms  and 
say  that  certain  diseases  are  communicable  from  man  to  man  or  from  animals 
to  man.  When  a  disease  picks  its  victims  rather  promiscuously,  in  a  cir- 
cumscribed area,  with  none  of  the  usual  characteristics  of  a  contagion  or 
infection,  we  usually  apply  the  term  epidemic.  For  example,  cerebro- 
spinal  meningitis  and  pneumonia  may  be  epidemical.  Diphtheria  is  often 


FIG.  77. — Bacillus  anthracis.  This  bacillus  is  spore-forming  and  causes  the  cattle 
disease  known  as  anthrax.  This  disease  is  especially  common  among  sheep  and  cattle 
and  may  be  transmitted  to  man,  especially  those  working  with  the  wool,  hides  and 
meat  of  infected  animals.  The  two  chief  forms  of  anthrax  in  man  are  malignant  pustule 
and  woolsorter's  disease.  The  dried  spores  of  this  bacillus  will  live  for  years  and  will 
withstand  the  boiling  temperature  for  hours.  Vaccinating  animals  against  anthrax  is 
commonly  practised  now.  Anthrax  is  frequently  confused  with  glanders,  an  equine 
disease  caused  by  the  Bacillus  mallei,  a,  Non-spore-bearing  bacilli;  b,  chains  of  cells;  c, 
spore-bearing  bacilli.  Cell-walls  and  plasmic  contents  are  stained,  the  spores  are  unstained. 

epidemic  in  a  community^  and  as  above  stated,  it  is  likewise  infectious  and 
contagious.  The  term  epidemic  is,  however,  also  applied  to  any  communi- 
cable disease  which  has  become  general  in  a  given  community.  A  more  or 
less  common  or  spreading  disease  which  is  limited  to  and  recurs  in  a  given 
district  or  country  is  said  to  be  endemic  in  that  district  or  country.  En- 
demics are  usually  due  to  climatic  conditions  which  encourage  certain 
microbic  and  other  disease-producing  invasions. 

The  causes  of  disease  are  of  two  kinds,  primary  or  inciting  and  secondary 
or  predisposing.  The  primary  cause  of  a  disease  is  that  factor  or  influence 
which  must  invariably  be  active  before  the  disease  can  possibly  develop. 
For  example,  the  primary  cause  of  diphtheria  is  the  diphtheria  bacillus;  the 
predisposing  causes  are  exposure  to  wet  and  cold,  impoverished  condition 


204 


PHARMACEUTICAL   BACTERIOLOGY. 


of  body,  etc.  No  matter  how  numerous  or  how  active  the  predisposing 
causes  may  be,  the  disease  cannot  develop  until  the  primary  cause  acts. 
There  are  numerous  abnormal  or  pathological  states  or  conditions  without 
recognizable  primary  causes,  as  gout,  rheumatism  and  the  senile  changes  in 
the  body:  and  again  there  are  certain  diseases  which  evidently  have  primary 
causes,  as  whooping-cough,  small-pox  and  yellow  fever,  but  in  which  said 
primary  causes  are  not  yet  discovered.  The  following  tabulation  outlines 
the  primary  and  secondary  causes  of  disease: 


Communicable 
diseases. 


Primary  causes 
(inciting). 


Bacteria,  as  in  typhoid  and  Asiatic  cholera. 

Protozoa,  as  in  malaria. 

Parasitic  higher  animals,  as  tape-worm  and 

itch. 

Fungi,  as  in  ring-worm  and  pellagra. 
Undetermined,   as  in    whooping-cough   and 

small-pox. 


Secondary  causes 
(predisposing). 


Heredity. . 


Age. 


Sex. 


Environment. 


Race. 

Family. 

Individual  (ontogenetic). 


(Phylogenetic). 


Habits. . . 


Infancy. 
Childhood. 
Adolescence. 
Adult. 
Old  age. 


Climate. 
Altitude. 
Seasons. 

Unsuitable  food. 
Unsuitable  clothing. 
Poisons. 
Occupation. 
Injuries. 

Alcoholic. 

Tobacco. 

Drugs. 

Coffee  and  tea. 

Gourmandage. 


In  a  general  way  it  may  be  stated  that  any  cause,  factor  or  influence, 
which  tends  to  lower  the  vitality,  predisposes  to  disease.  Individuals  with 
a  well-balanced  physical  and  mental  development  are  less  liable  to  disease, 
and  when  attacked  are  more  apt  to  recover,  than  those  individuals  who 
have  a  poor  physical  development.  Undue  abstinence  is  as  harmful  as 
over-indulgence.  The  ascetic  is  as  pathologic  as  the  gouty  gourmand. 


COMMUNICABLE  DISEASES.  205 

Irrational  diet,  drink  and  food  fads,  sooner  or  later  leave  their  pernicious 
effects  upon  the  system  and  predispose  to  certain  diseases.  Overeating 
is  as  objectionable  as  starvation.  Lack  of  adequate  physical  exercise  has 
its  evil  effects  as  does  also  over-exertion.  Trained  or  professional  athletes 


/  / 


IS  ^  >> 


FIG.  78.  FIG.  79. 

FIG.  78. — Bacillus  mallei,  the  cause  of  glanders  in  horses.  This  disease  can  be  trans- 
mitted to  man  where  it  causes  symptoms  of  a  suppurative  infection  of  the  lymphatic  glands. 
Mallein,  which  is  used  in  testing  horses  for  glanders,  consists  of  the  nitrate  (Berkefeld 
filter)  of  dead  cultures  (glycerin  bouillon)  of  the  bacillus.  A  positive  malleiu  reaction 
consists  in  a  rise  in  temperature  and  local  swelling.  The  dose  is  i  c.c. 

FIG.  79. — Bacillus  tetani,  an  anaerobic  spore-bearing  bacillus,  the  cause  of  tetanus  or 
lockjaw.  This  bacillus  is  found  in  soils  and  may  infect  abrasions,  cuts  and  wounds. 
Treatment  with  tetanic  antitoxin  is  successful  if  begun  before  the  symptoms  develop.  The 
best  time  to  administer  the  antitoxin  is  at  the  time  the  injury  is  received. 


FIG.  80. — A  spore-bearing  bacillus  stained  with  methyl  blue  leaving  the  spores  unstained. 
Fortunately  most  of  the  bacilli  pathogenic  to  man  do  not  bear  spores. 

are  not  long  lived,  many  are  hopelessly  afflicted  with  enlarged  and  weakened 
heart  and  arteries  (aneurism).  Pernicious  habits  of  all  kinds  indicate 
weakness  and  further  develop  the  weakness,  which  in  turn  predisposes  to 
certain  diseases  and  render  the  individual  less  resistant  to  the  ravages  of 
disease.  A  good  ancestry  and  inheritance,  good  wholesome  food,  comfort- 


206  PHARMACEUTICAL   BACTERIOLOGY. 

able  clothing,  the  right  sort  of  exercise  for  body  and  mind,  the  simple  life 
rather  than  the  strenuous  life,  avoiding  bad  habits  of  all  kinds,  abundant 
fresh  air,  etc.,  all  tend  toward  longevity.  To  argue  that  we  should  go  un- 
clothed is  as  absurd  and  unreasonable  as  to  teach  that  sheep  should  be 
shaved.  To  adhere  to  a  wholly  vegetable  diet  is  irrational  simply  because 
we  are  organically  adapted  to  a  mixed  diet.  An  excessive  meat  diet  is  also 
very  pernicious. 

Occupation  is  a  potent  factor  in  predisposing  to  disease,  and  in  lon- 
gevity. The  following  table  adapted  from  a  report  by  Ogle  will  serve  to 
make  this  clear.  The  high  mortality  rate -among  street-hawkers  is  due  to 
several  causes  chief  of  which  are  low-living,  exposure  to  inclement  weather, 
and  the  greater  exposure,  in  the  squalid  districts  of  large  cities,  to  the  primary 
causes  of  disease.  The  low  mortality  rate  among  clergymen  is  due  to  a 
comparatively  simple  though  comfortable  mode  of  living;  while  in  the  case 
of  the  farmer  and  gardener,  the  out-of-door  life  is  the  favorable  influence. 
The  list  represents  ages  ranging  from  twenty-five  to  sixty  years,  therefore 
adults. 

Occupation  Comparative 

Mortality 

Clergymen,  priests  and  ministers 100 

Gardeners 108 

Fanners 114 

Carpenters 147 

Lawyers 152 

Coal  miners 160 

Bakers 172 

Builders,  masons,  bricklayers 174 

Blacksmiths 175 

Commercial  clerks 179 

Tailors 189 

Cotton  manufacturers 196 

Medical  men 202 

Stone,  slate  quarries 202 

Book-binders 210 

Butchers 211 

Glass  workers 214 

Plumbers,  painters,  glaziers 216 

Cutler,  scissors  makers 229 

Brewers 245 

Innkeeper,  liquor  dealers 274 

File  makers 300 

Earthenware   workers 314 

Street  hawkers 338 

Inn,  hotel  service 396 

The  following  are  the  more  important  communicable  diseases  with 
suggestions  on  prevention.  The  information  is  given  for  the  sole  purpose 


COMMUNICABLE    DISEASES.  207 

to  better  qualify  the  pharmacist  to  cooperate  with  the  health  officers  in 
safeguarding  the  public  health. 

A.  Tuberculosis. — Commonly  known  as  consumption  and  the  " white 
plague."  A  universal  disease,  essentially  infectious,  especially  peculiar  to 
crowded  habitations  and  to  lack  of  pure  fresh  air.  The  primary  cause  is 
the  Bacillus  tuberculosis  (bacillus  of  Koch),  a  non-spore-bearing  microbe, 
which  is  somewhat  more  resisting  to  disinfectants  and  other  destructive 
agencies  than  most  other  pathogenic  bacteria.  The  chief  predisposing 
causes  are  living  in  crowded  habitations;  inherited  low  vitality,  especially 
weak  lungs;  and  exposure  to  inclement  weather.  The  disease  may  be 
general  (general  tubercular  infection)  or  it  may  be  localized  in  any  one  or  in 
several  organs  or  tissues.  Commonly  localized  in  the  lungs  (phthisis,  con- 
sumption) and  in  lymph  glands.  Lupus  and  many  so-called  scrofulous 
conditions  are  tuberculosis  of  the  skin;  the  disease  often  attacks  bones  and 


f  -. 


IN 

FIG.  81. — Bacillus  tuberculosis.  Although  this  organism  does  not  form  spores  it  is 
quite  resistant  to  the  action  of  germicides.  The  bacillus  causing  the  bovine  type  of  tuber- 
culosis differs  slightly  in  several  characteristics  from  the  bacillus  of  human  tuberculosis. 

joints  (hip-joint  disease  of  children).  It  attacks  young  and  old  and  may 
occur  in  all  walks  of  life.  The  disease  enters  via  the  air  passages  and  per 
mouth  with  food  and  drink,  or  through  cuts,  bruises,  wounds  and  abrasions. 
It  is  contracted  by  inhalation  through  close  association  with  consumptives, 
and  the  bovine  form  or  type  of  tuberculosis  is  acquired  from  the  milk  of 
tubercular  cows.  Bovine  tuberculosis  is  especially  liable  to  affect  the  lymph 
glands  and  the  joints. 

The  disease  sometimes  runs  a  quick  course  (quick  consumption),  but 
more  generally  it  makes  an  insidious  start  and  runs  a  chronic  course.  Many 
people  have  limited  local  infections  which  are  only  discovered  at  an  autopsy. 
There  are  many  spontaneous  recoveries  from  tuberculosis.  Since  it  is 
very  important  to  begin  early  treatment,  the  physician  resorts  to  several 
tests  for  the  purpose  of  determining  the  possible  existence  of  masked  or 
incipient  forms  of  the  disease.  These  tests  are  as  follows  and  all  depend 


2O8  PHARMACEUTICAL   BACTERIOLOGY. 

upon   the   reactions   produced   by  tuberculins   when  introduced  into   the 
system: 

a.  The  Calmette  or  Ophthalmo  Test. — Old  tuberculin,  precipitated  by 
alcohol  is  used.     The  precipitate  is  dried  and  made  into  a  i  per  cent,  solu- 
tion in  sterilized  distilled  water  or  sterile  physiologic  salt  solution.     This 
substance  is  put  up  in  sterile  capillary  pipettes,  ready  for  use.     A  drop  of 
the  solution  is  placed  in  one  eye,  using  the  other  eye  as  a  control.     Any 
abnormality  in  the  eye  is  regarded  as  a  contraindication.     If  tuberculosis 
exists  in  the  system  it  is  indicated  by  an  inflammation  in  the  eye  tested. 
Also  known  as  the  Wolff-Eisner  test  or  reaction.     It  may  be  necessary  to 
repeat  the  test  several  times  before  satisfactory  results  are  obtained. 

b.  The  von  Pirquet  or  Cutaneous  Test. — A  25~per  cent,  solution  of  tuber- 
culin (O.  T.)  is  applied  to  the  skin  with  scarification,  as  in  vaccination. 
The  skin  is  first  cleansed  with  alcohol  and  control  scarifications  are  made 
near  the  test  area.     This  test  is  also  known  as  the  "skin  reaction."     It  is 
not  very  reliable.     The  inflammatory  reaction  may  be  simulated  by  other 
substances  in  persons  that  are  known  to  be  entirely  free  from  tuberculosis. 

c.  The  Moro,  Percutaneous  or  Ointment  Test. — Fifty  per  cent,  tuberculin 
(O.  T.)  in  lanolin  is  rubbed  into  the  skin,  without  scarification.     The  prep- 
aration is  put  up  in  collapsible  tubes,  one  tube  containing  enough  material 
for  several  tests.     If  tuberculosis  exists,  small  reddened  vesicles  appear  at 
the  point  of  inunction,  usually  on  the  second  day. 

d.  The  Thermal  Test. — A  solution  of  tuberculin  (O.  T.),  put   up   in 
8  c.c.  bottles,  representing  one  milligram  per  c.c.  (i-iooo)  is  injected  hypo- 
dermically.     If  tuberculosis  is  present  there  is  a  rise  in  temperature,  usually 
within  ten  to  twenty-four  hours  after  injection. 

e.  The  Detre  Differential   Test. — This  test  is  intended  to  differentiate 
between  tuberculosis  of  human  origin  and  that  of  bovine  origin.     Three 
tuberculins  are  required.     Tuberculin  O.  T.,  tuberculin  B.  F.,  made  from 
tubercle  bacilli  of  human  origin  and  tuberculin  B.  F.,  made  from  tubercle 
bacilli  of  bovine  origin.     Three  small  skin  areas  are  scarified.     Into  one 
tuberculin  O.  T.  is  rubbed,  into  the  second  humanized  tuberculin,  and  into 
the  third  bovinized  tuberculin.     The  resulting  reactions  indicate  whether 
tuberculosis  is  of  human  or  cf  bovine  origin. 

We  cannot  go  into  the  details  of  the  reactions.  They  are  not  always 
reliable,  neither  the  positive  nor  the  negative  reactions.  In  the  advanced 
stages  of  tuberculosis  and  in  moribund  cases,  the  reaction  is  usually  negative. 
.  Indeed,  in  such  cases  the  test  is  unnecessary  as  the  existence  of  the  disease 
is  evident  without  special  tests. 

Tuberculosis  is  not  as  infectious  as  is  generally  supposed.  Those  who 
are  in  good  condition  physically  may  live  for  years  with  those  afflicted  with 
the  disease  without  becoming  infected.  Yet,  tubercular  patients  should  be 


COMMUNICABLE   DISEASES.  209 

isolated  from  well  people  as  much  as  possible.  The  sputum  is  the  principal 
source  of  infection,  also  other  secretions;  and  the  breath  as  in  sneezing,  laugh- 
ing and  coughing.  Plenty  of  fresh  pure  dry  air  should  be  supplied  to  patients, 
large  airy  sleeping  rooms  and  easily  digested  wholesome  food  is  essential. 
Consumptives  should  not  marry,  should  not  kiss  healthy  individuals,  espe- 
cially children.  Expectorated  material  should  be  disinfected  at  once.  Treat- 
ment should  be  begun  early.  The  propaganda  favoring  well  constructed, 
well  ventilated,  comfortably  warmed  homes  and  less  close  segregation  in 
cities  and  a  general  improvement  in  sanitation  will  do  much  toward  eradi- 
cating tuberculosis.  Tenement  houses  and  large  or  small  crowded  houses 
.of  all  kinds  should  not  be  tolerated  for  moral  as  well  as  for  sanitary  reasons. 
Above  all,  see  to  it  that  the  milk  used  is  free  from  tubercular  infection. 

B.  Typhoid  Fever. — This  is  a  filth  disease.  If  the  environment  were 
made  clean  and  sanitary,  typhoid  fever  could  not  exist.  The  primary  cause 
is  the  non-sporulating  Bacillus  typhosis  which  is  found  in  filthy  water,  in 
milk  and  in  food  materials.  Slops,  sewage,  wash  water,  etc.,  poured  on  the 
soil  may  seep  into  the  well  water  and  finally  enter  the  system  in  drinking. 
The  bacillus  develops  readily  in  the  intestinal  tract  where  the  reaction  is 
alkaline.  It  is  quite  susceptible  to  the  action  of  weak  acids  and  is  easily 
killed  by  boiling  and  by  disinfectants.  Typhoid  is  a  widely  disseminated 
dangerous  infectious  as  well  as  contagious  disease.  In  large  cities  the 
mortality  rate  from  this  disease  is  directly  proportional  to  the  filthiness  of 
the  drinking-water  supply.  In  country  districts  epidemics  are  very  fre- 
quently due  to  contaminated  well-water  (contaminated  from  kitchen  refuse, 
barns,  cow-sheds,  etc.).  Epidemics  often  follow  in  the  wake  of  the  dairy- 
man, who  supplies  cow's  milk  in  cans  washed  with  or  which  contain  milk, 
adulterated  with  polluted  water.  Typhoid  fever  is  carried  in  vegetables  from 
truck  gardens  where  human  and  other  excrement  are  used  for  fertilizing 
purposes.  The  Chinese  truck  gardeners  are  particularly  culpable  in  this 
regard.  Again,  the  vegetables  are  irrigated  with  stagnant  and  sewage- 
polluted  water.  House  flies  are  carriers  of  typhoid. 

The  mortality  rate  in  typhoid  is  high  and  the  disease  runs  its  course  in 
about  five  weeks.  There  are  some  mild  cases,  the  so-called  walking  or 
ambulatory  cases.  All  of  the  excreta  from  the  patient  should  be  disin- 
fected, using  corrosive  sublimate  solution  (i-iooo),  copperas  solution 
(10  to  20  per  cent.),  blue  vitriol  solution  (5  to  15  per  cent.),  milk  of  lime 
(for  stools),  etc.  All  bed  linen,'  clothing,  etc.,  used  by  the  patient  should  be 
disinfected  in  5-per  cent,  carbolic  acid  before  washing.  Everything  used 
by  the  patient  should  be  sterilized,  disinfected  and  kept  away  from  the  rest 
of  the  family.  Those  who  nurse  typhoid  patients  must  be  extremely  care- 
ful not  to  carry  the  infection  to  others.  Pillows,  mattresses  and  other  large 
articles  used  by  the  patient  should  be  steam  sterilized,  or  if  that  cannot  be 
14 


210  PHARMACEUTICAL   BACTERIOLOGY. 

done  conveniently,  they  should  be  destroyed  by  burning.  In  simple  words, 
everything  about  the  patient  must  be  scrupulously  sterilized  in  order  to 
avoid  spreading  the  infection. 

A  national  department  of  health  should  see  to  it  that  the  water  supply  of 
large  cities  is  free  from  sewage  contamination.  Our  streams,  lakes  and 
reservoirs  supplying  drinking  water  require  careful  guarding  against  typhoid 
infection. 

There  should  be  a  compulsory  regulation  regarding  the  position  and 
depth  of  wells  in  farm  yards  and  as  regards  the  position  of  the  well  relative 

to  barns,  cow  sheds,  privy  vaults,  etc.     Typhoid 
^    0     ^   -  fever  will  continue  its  ravages  as  long  as  filth 

^      ^v  ^  ^  contamination  of  water  supplies  and  food  sup- 

\    £*    &^>     0    #      plies  is  permitted. 

«=»      ^  ^  f\  The    Gruber-Widal   test   for   typhoid  is  an 

4=9    ^  *=>        ^      agglutination  phenomenon.     The  agglutinating 

^       ff  0     ^     ^  f)     power  of  the  blood  of  a  typhoid  patient  is  usually 

^   0   /)  0<=3 ==>  noticeable  as  early  as  the  fifth  day  of  the  dis- 

<^  ^        *^>v^       ease-     Preventive  inoculation  with  typhoid  bac- 

^      ^  j)          terin  has  been  used  with  considerable  success, 

particularly  in  the  British  and  German  armies, 
Fig.     82. — Bacillus    pneu-         ,    •  .,  ,  ,    . 

monia  of  Friedlander,  also  and  IS  now  <lmte  extensively  used  in  general 
known  as  Bacillus  mucosus.  practice.  Chantamesse  and  Wright  use  agar 

or  broth  cultures  of  the  typhoid  bacillus,  killed 
by  heat.  It  is  declared  by  some  physicians  that 
the  bacterin  administered  early  in  the  disease,  checks  it,  and  occasionally 
effects  a  prompt  cure. 

C.  Pneumonia. — Pneumonia  with  its  modifications,  as  broncho-pneu- 
monia, capillary  bronchitis,  pleuro-pneumonia,  pneumonic  pericarditis,  etc., 
is  extremely  common.  The  primary  cause  is  the  non-sporogenous  Diplo- 
coccus  pneumonia  (Micrococcus  lanceolatus) .  The  important  predisposing 
causes  are  exposure  to  wet  and  cold,  weak  lungs,  infancy,  old  age,  general 
debility  and  alcoholism.  The  disease  is  not  very  infectious,  in  fact  is  not 
generally  so  considered.  It  is  generally  limited  to  the  respiratory  tract  and 
the  contiguous  tissues,  as  the  pericardium  and  the  pleurae.  Among  infants 
and  young  children  and  those  well  past  middle  life,  the  disease  shows  a 
high  mortality  rate.  In  youth  and  early  middle  life  recovery  is  the  rule, 
provided  the  physical  inheritance  and  development  is  good.  The  mortality 
rate  among  those  addicted  to  the  use  of  alcoholic  drinks,  and  those  affected 
with  " tobacco  heart,"  is  very  high. 

One  attack  of  pneumonia  is  supposed  to  increase  the  resisting  power  to 
subsequent  attacks  but  such  acquired  immunity  is  not  by  any  means  per- 
manent. The  anti-pneumococcic  serum  is  used  with  some  apparent  success 


COMMUNICABLE   DISEASES.  211 

though  the  results  are  far  from  satisfying  to  the  majority  of  those  who  have 
tried  it.  Dr.  Shafer  has  recently  recommended  a  mixed  bacterin  (com- 
posed of  disease  exudate  and  pure  pneumonic  bacterin)  which  has  been  used 
with  some  success. 

It  is  important  to  guard  against  exposure  to  wet  and  cold,  particularly 
when  the  vitality  of  the  body  is  lowered,  as  through  lack  of  sleep,  lack  of 
food,  over-exertion,  etc.  The  sputa  of  patients  should  be  disinfected  at 
once.  Well  persons  having  good  resisting  power  may  carry  the  germs  and 
convey  the  disease  to  those  who  have  a  lower  vitality.  The  room  occupied 
by  the  patient  should  be  thoroughly  fumigated  as  soon  as  possible. 

D.  Small -pox. — Also  known  as  variola  and  pest.  This  is  a  well-known 
disease  which  has  occurred  epidemically  from  time  to  time  throughout  all 
ages  and  in  all  lands.  It  is  most  highly  infectious  and  contagious.  In  spite 
of  all  investigations,  the  primary  cause  has  not  yet  been  discovered.  The 
contagion  is  carried  by  the  breath  of  the  patient,  is  wafted  from  the  skin 
eruptions,  is  carried  in  clothing  and  by  everything  used  or  touched  by  the 
patient.  The  contagion  may  lie  dormant  in  clothing  for  months. 

All  excreta  from  the  patient  should  be  disinfected  with  bichloride  of 
mercury  (i-iooo)  or  a  5  per  cent,  solution  of  carbolic  acid  or  other  con- 
venient disinfecting  agents  as  lime,  formalin,  etc.  Bedding,  mattresses  and 
other  material  used  in  the  sick-room  should  be  burned  as  soon  as  the  patient 
does  not  need  them  any  longer. 

As  the  result  of  the  general  practice  of  vaccination  (with  the  modified 
cow  virus)  small-pox  is  no  longer  the  dread  disease  that  it  once  was.  In 
Germany,  where  vaccination  is  carried  out  with  greatest  strictness,  the 
mortality  rate  from  small-pox  is  about  o.i  per  cent.  In  the  United  States 
where  vaccination  is  laxly  enforced,  the  mortality  rate  is  about  3  per  cent 
Since  vaccination  is  almost  an  absolute  safeguard,  there  is  no  need  of  fear- 
ing this  disease,  even  when  brought  in  direct  contact  with  it  One  vacci- 
nation does  not  establish  life  immunity,  as  is  popularly  believed.  The  rule 
is  to  vaccinate  in  infancy,  again  about  the  time  of  adolescence  and  again 
in  early  adult  life.  This  will  usually  insure  immunity  for  life.  However, 
vaccination  should  be  carried  out  after  every  exposure  or  whenever  small- 
pox exists  in  the  vicinity,  no  matter  how  many  good  "take"  scars  there 
may  be.  Nurses  and  physicians  in  pest  hospitals  are  vaccinated  once  a 
year,  or  of tener,  to  insure  immunity.  In  the  navy  it  is  customary  to  vac- 
cinate every  man  every  time  a  port  is  entered  where  small-pox  is  suspected. 
Small-pox  is  a  quarantinable  disease. 

There  is  absolutely  no  danger  or  ill  effect  from  vaccination,  in  spite  of  the 
popular  newspaper  and  popular  verbal  reports  to  the  contrary.  In  perhaps 
one  case  in  a  million,  tetanus  or  severe  septicemia  may  be  traceable  to  the 
use  of  an  impure  virus.  Septic  infection  of  the  scarified  area  may  take 


212  PHARMACEUTICAL   BACTERIOLOGY. 

place,  due  to  carelessness  on  the  part  of  the  patient,  and  not  due  to  the 
virus  used,  but  even  this  is  an  extremely  rare  occurrence.  Since  the  incu- 
bation period  of  small-pox  is  about  twelve  days  and  that  of  vaccinia  (cow- 
pox)  is  only  five  or  six  days,  it  is  evident  that  the  vaccination  will  establish 
immunity  even  in  those  who  were  actually  exposed,  provided  vaccination 
is  done  within  a  few  days  aften  exposure. 

Primitive  (savage)  races  are  very  susceptible  to  small-pox,  with  a  very 
high  mortality  rate.  This  is  in  part  due  to  the  total  ignorance  of  sanitary 
measures,  resulting  in  the  more  ready  spread  of  the  contagion.  Entire 
savage  tribes  have  been  exterminated  by  this  disease.  Negroes  are  far 
more  susceptible  than  Caucasians.  Indians  have  spread  the  infection  in 
blankets  after  having  been  exposed. 

E.  Malaria. — This  familiar   disease,  commonly  known   as  ague,  the 
shakes,  chills  and  fever,  and  intermittent  fever,  prevails  in  many  areas  in 
the  United  States  and  is  limited  to  swampy  wet  countries.     It  gradually 
disappears  with  the  tilling  and  the  draining  of  soil  which  remove  the  breed- 
ing places  of  the  only  carriers  of  the  disease,  namely  the  mosquitos   (An- 
opheles).    The  primary  causes  is  the  Plasmodium  malaria  (Hcematozoa 
malaria}  which  is  introduced  into  the  circulation  by  the  sting  of  the  mos- 
quito. 

The  prophylactic  measures  consist  in  the  destruction  of  the  mosquitos 
in  rooms.  To  this  end  burn  two  pounds  of  Pyrethrum  to  every  thousand 
cubic  feet  of  space.  Sulphur  one  pound  per  thousand  cubic  feet  may  be 
used  though  it  offers  no  advantage  over  the  Pyrethrum  and  has  the  dis- 
advantage of  corroding  metal  and  fading  colored  fabrics.  Also  destroy  the 
breeding  places  of  the  mosquito  and  keep  mosquitos  out  of  houses  by  means 
of  screens  and  netting.  Protect  the  person  against  mosquito  stings  when 
travelling  in  countries  known  to  be  infested  by  the  Anopheles  group  of 
mosquito.  Also  take  quinine  as  a  prophylactic  (3  to  5  grains  twice  daily), 
and  as  a  cure.  Quinine  is,  however,  more  satisfactory  as  a  preventive  than 
as  a  cure.  The  Plasmodium  is  known  to  be  very  susceptible  to  the  action 
of  quinine. 

F.  Diphtheria. — This  dread  disease  is  both  infectious  and  contagious. 
The  primary  cause  is  the  Bacillus  diphtheria  also  known  as  the   Klebs- 
Loeffler  bacillus.     The  chief  predisposing  causes  are  exposure  to  wet  and 
cold.     The  disease  may  be  localized  in  the  larynx  (membranous  croup),  in 
the  pharynx,  in  the  nares,  on  any  of  the  mucous  membranes,  and  in  cuts 
and  wounds.     Animals  such  as  cats  may  carry  the  infection.     It  is  also 
stated  that  the  bacillus  is  apt  to  occur  in  certain  soils  and  in  stable  manure. 
The  sick  must  be  isolated  and  all  discharges  from  nose,  mouth  and  throat 
as  well  as  the  bed  linen,  etc. ,  must  be  sterilized  and  disinfected.     Upon  recovery 
the  sick-room  must  be  thoroughly  fumigated  by  means  of  formaldehyde. 


COMMUNICABLE   DISEASES.  213 

Bedding,  mattress  and  pillows  should  be  burned.  The  anti-diphtheric  serum 
should  be  used  early  and  in  large  doses.  The  best  authorities  look  upon 
this  remedy  as  a  specific,  always  effecting  a  cure,  provided  it  is  given  in  time 
and  given  in  adequate  doses.  All  those  who  have  been  exposed  should 
receive  a  prophylactic  dose  of  the  remedy  (about  500  units).  The  other 
remedial  agents  as  gargles,  sprays,  etc.,  should  not  be  neglected.  The 
diphtheria  toxin  acts  on  the  heart  and  all  patients  should  be  warned  against 
any  sudden  or  severe  exertion  until  complete  recovery  is  assured  by  the 
attending  physician  as  death  has  resulted  from  a  single  undue  action,  as 
jumping  or  suddenly  rising  from  bed. 

G.  Cancer. — The  primary  cause,  the  secondary  cause  and  the  treat- 
ment of  cancer  are  all  in  the  dark  as  yet.  We  know  that  this  disease  rarely 
develops  earlier  than  middle  life.  It  usually  runs  a  comparatively  short 
course  (several  months  to  two  years),  producing  some  rather  marked  symp- 
toms (the  cancerous  cachexia),  with  constant  pain,  and  a  very  characteristic 
waxy  pallor  of  the  skin.  It  is  to  be  hoped  that  the  primary  cause  and  the  cure 
will  be  discovered  in'  a  short  time.  There  are  some  indications  that  a 
tendency  to  cancer  is  inherited  and  that  the  primary  cause  is  an  organism 
resembling  the  protozoa  group.  There  is  a  popular  belief  that  eating  raw 
tomatoes  causes  cancer,  and  it  may  be  that  the  plasmodium  of  cancer 
resides  in  some  vegetable.  Cancer  may  attack  any  tissue  or  organ,  although 
the  internal  viscera,  as  liver  and  stomach,  are  more  commonly  affected. 
Cancer  should  be  treated  as  a  contagious  disease  though  the  proof  of  its 
contagious  nature  is  not  conclusive. 

All  advertised  cancer  cures  are  fakes.  There  is  no  known  cure  for  can- 
cer, unless  the  Oilman  cancer  vaccine  proves  to  be  one.  Surgical  removal  of 
cancerous  growths  has  been  the  means  of  prolonging  life,  but  the  trouble  is 
very  apt  to  recur.  Many  cases  are  inoperable. 

H.  Plague. — This  disease,  which  is  also  known  as  black  plague,  the  pest 
bubonic  plague,  black  death,  etc.,  is  essentially  a  filth  disease.  The  pri- 
mary cause  is  the  non-sporogenous  Bacillus  pestis.  The  plague  has  occurred 
epidemically  from  time  to  time  throughout  all  ages.  It  is  most  virulent  and 
most  prevalent  (endemic)  in  the  crowded  cities  of  the  warmer  countries 
(Oriental  cities),  where  the  sanitary  conditions  are  often  very  bad.  The 
disease  is  highly  contagious  and  infectious  and  is  communicable  not  only  to 
man  but  also  to  rats,  mice,  dogs,  squirrels  and  cattle.  Rats,  and  the  fleas 
upon  them,  are  the  principal  carriers  of  the  disease,  although  other  animals 
as  ants  and  flies  may  also  act  as  carriers. 

There  are  several  forms  of  the  plague  of  which  the  pneumonic  is  the  most 
dangerous  and  most  infectious  because  the  bacilli  are  spread  by  coughing 
and  sneezing. 

In  this  disease  thorough  disinfection  is  of  the  greatest  importance.     The 


214  PHARMACEUTICAL    BACTERIOLOGY. 

entire  body  of  the  patient  should  be  washed  with  a  disinfecting  solution 
(1-1200  bichloride  of  mercury).  Disinfect  everything  used  about  the 
patient.  After  death  or  recovery  everything  used  by  the  patient  should  be 
destroyed  by  burning. 

Rats  (bearing  the  infected  fleas)  are  the  principal  carriers  of  this  disease, 
and  the  experience  in  San  Francisco  (1906-1909)  has  demonstrated  that 
plague  disappears  as  soon  as  the  plague  infested  rats  disappear.  Destroy 
rats  and  mice  and  see  to  it  that  the  home  is  free  from  fleas.  Plague  is  a 
quarantinable  disease  and  the  federal  authorities  are  constantly  on  the  look- 
out to  prevent  the  importation  of  this  disease.  The  Oriental  ports  are  the 
chief  sources  of  infection. 


. 


FIG.  83.  FIG.  84. 

FIG.  83. — Bacillus  pestis.  Does  not  form  spores  and  is  very  easily  killed.  The  ends 
stain  more  heavily  than  the  middle.  Involution  forms  may  accur.  Sometimes  the  cells 
become  encapsuled  as  shown  in  the  figure. 

FIG.  84. — Bacillus  cholera  also    known  as  Spirillum    choleras,    the  cause  of  Asiatic 

cholera. 

Yersin's  anti-plague  serum  and  Haffkine's  bacterin  have  been  used  with 
considerable  success  as  prophylactics  and  also  with  some  success  as  cures. 

I.  Asiatic  Cholera. — This  is  another  filth  disease  essentially  of  Oriental 
origin,  particularly  prevalent  in  the  crowded  unsanitary  cities  of  India  and 
Asia.  It  is  a  quarantinable  disease.  The  primary  cause  is  the  non-spor- 
ogenous  Bacillus  cholera  (Spirillum  cholera)  also  known  as  the  comma 
bacillus  of  Koch.  The  principal  sources  of  the  infection  are  polluted  water 
and  food,  particularly  the  former.  In  fact  the  sources  of  infection  and  modes 
of  entry  into  the  digestive  tract  are  not  unlike  those  of  typhoid.  Cholera  is 
highly  infectious  and  usually  occurs  epidemically,  often  spreading  over  wide 
areas.  Human  excrement  carries  the  infection  and  when  this  material  is 
used  as  fertilizer,  which  is  done  in  China  and  other  Oriental  countries,  it 
becomes  the  means  of  initiating  and  continuing  the  spread  of  the  disease. 
The  importing  by  the  Chinese  of  human  excrement  and  animal  dung  for 


COMMUNICABLE   DISEASES.  215 

medicinal  purpose  should  be  prohibited  as  it  may  be  the  means  of  starting 
an  epidemic  of  cholera  in  the  United  States. 

Fortunately  the  cholera  bacillus  is  easily  killed  by  heat,  disinfectants,  and 
by  drying.  The  temperature  of  boiling  water  kills  it  in  five  minutes.  In 
water  it  may  retain  its  vitality  for  a  long  time.  Furthermore,  it  is  not  a  strict 
(obligative)  parasite  and  may  multiply  outside  of  the  body  under  favorable 
conditions.  Flies  carry  the  infection  from  cholera  stools  to  articles  of  food. 

Haffkine's  attenuated  cholera  bacterin  has  been  employed  successfully 
as  a  prophylactic.  The  method  of  use  consists  first  in  the  hypodermic  in- 
jection of  a  weak  virus,  that  is,  cultures  attenuated  by  long  cultivation  at  a 
high  temperature  (39°  C.),  and  following  this  later,  in  five  days,  with  a 
virulent  culture.  More  recently  Kolle  has  used  cultures  killed  by  heating 
for  one  hour  at  58°  C.,  which  has  given  good  results  in  numerous  tests  made 
during  a  cholera  epidemic  in  Japan.  Pfeiffer  and  others  have  experimented 
extensively  with  cholera-immune  serum  and  have  demonstrated  that  this 
has  marked  lytic  properties.  The  cholera  bacilli  when  placed  into  the  serum 
first  lose  motility,  then  swell  up  into  coccus-like  forms  and  finally  dissolve. 
This  property  is  said  to  be  due  to  two  substances,  one  found  in  normal  serum 
and  the  other  found  in  immune  serum.  Neither  substance  alone  can  de- 
stroy the  cholera  bacilli  but  the  two  acting  together  are  strongly  bacterio- 
lytic.  The  immunity  produced  by  the  Haffkine  and  Kolle  bacterins  is 
temporary  only. 

J.  Yellow  Fever. — This  highly  infectious,  but  in  no  wise  contagious, 
disease  is  peculiar  to  tropical  and  subtropical  countries.  The  primary 
cause  is  as  yet  unknown  but  it  is  supposed  to  be  a  protozoan.  The  sole 
carrier  of  the  infection  is  a  mosquito,  Stegomyia  calopus.  The  disease  has 
been  highly  epidemical  in  the  southern  states  but  since  the  discovery  of  the 
part  played  by  the  mosquito  the  mortality  rate  has  been  lowered  to  a  marked 
degree.  In  fact  the  disease  is  now  under  complete  control.  No  Stegomyia 
mosquitos,  no  yellow  fever. 

It  had  been  observed  for  a  long  time  that  a  frost  checked  the  disease  at 
once,  which  as  is  now  known,  was  due  to  the  fact  that  the  frost  killed  the 
carriers  of  the  infection.  In  a  general  way  the  statements  made  under 
malaria  prophylaxis  also  apply  here.  Caucasians,  especially  those  not 
acclimated  in  the  yellow-fever  countries,  are  very  susceptible  to  the  disease; 
Negroes  and  Latin  races  are  far  less  susceptible. 

K.  Pellagra. — Pellagra  is  a  disease  which  has  created  great  havoc  in 
Italy  and  other  Eastern  countries,  and  which  first  appeared  in  the  United 
States  about  1907.  It  spread  very  rapidly  and  up  to  1911  numerous  cases 
have  been  reported  from  the  Southeastern  United  States  and  from  Illinois, 
with  a  few  scattering  cases  from  Kansas,  Virginia,  Pennsylvania,  New 
York,  Massachusetts,  California  and  other  states.  The  disease  is  said  to 


2l6  PHARMACEUTICAL   BACTERIOLOGY. 

be  caused  by  eating  mouldy  corn  (Zea  mays)  or  foods  prepared  from  such 
corn.  Ceni  and  others  declare  that  the  primary  cause  is  a  species  of  As- 
pergillus  (A .  flavescens  and  perhaps  also  A .  fumi gatus) .  It  is  also  believed 
that  the  ordinary  household  mould  (Penicillmm  glaucvm)  is  a  primary  cause. 
The  mortality  rate  is  very  high,  and  the  disease  is  said  to  be  terrible  in  its 
effects.  It  first  manifests  itself  as  an  eruption  of  the  skin,  usually  appearing 
in  the  early  spring,  February  or  March,  after  some  variable  prodromal  symp- 
toms. The  skin  becomes  darkened  and  blotchy.  Eczematous  eruptions 
next  appear,  with  desquamation.  Gradually,  as  the  older  eruptions  heal, 
while  new  ones  form,  the  skin  becomes  rough,  from  which  the  name,  pell'  agra 
— rough  skin — is  derived.  The  symptoms  increase  from  year  to  year. 
The  nervous  manifestations  are  varied  and  are  accompanied  by  great 
suffering. 

Pellagra  is  not  contagious  or  infectious,  though  the  tendency  is  trans- 
mitted from  one  generation  to  another.  Children  of  pellagrins  are  often 
born  with  asymmetrical  heads  and  various  other  deformities.  They  may 
be  idiotic  or  stupid  and  defective  generally. 

Acute  pellagra  runs  a  rapid  course,  but  more  generally  it  is  chronic,  the 
suffering  continuing  for  years  in  an  ever  increasing  ratio.  The  sufferers 
simply  degenerate  from  year  to  year  and  die  a  slow  but  terrible  death. 

Lombrosa,  Ceni  and  others  recognized  the  fact  that  pellagrins  are  mostly 
of  the  poorer  class,  whose  principal  diet  is  polenta,  a  mush  made  from 
corn  meal.  This  mush  is  usually  prepared  in  large  potfuls,  sufficient  for  a 
week's  eating,  and  set  away,  exposed  to  dust,  dirt,  flies,  etc.,  so  that  these 
ignorant  peasants  often  eat  polenta  which  is  more  or  less  mouldy  and  other- 
wise spoiled.  Efforts  were  at  once  made  to  correct  these  conditions,  but 
proved  only  partially  successful  as  far  as  checking  the  ravages  of  the  disease 
was  concerned. 

L.  Syphilis. — This  is  a  filth  disease  of  which  the  primary  cause  is  the 
Spiroch&ta  pallida,  though  several  other  organisms  are  generally  found 
present,  which  have  also  been  designated  as  being  causative  of  the  disease. 
This  is  believed  to  be  the  most  widely  disseminated  disease  of  civilization. 
It  is  essentially  chronic  in  its  course,  the  effects  being  apparent  even  in 
the  third  and  fourth  generations.  Primitive  races  are  said  to  have  been 
free  from  this  disease  until  the  advent  of  civilization,  yet  the  disease  is  of 
great  antiquity  having  been  widespread  in  ancient  Rome  and  Greece.  It 
is  very  infectious  via  abrasions,  cuts  and  all  breaks  in  the  continuity  of  the 
skin  and  mucous  membranes.  The  infection  is  carried  by  all  manner  of 
exposed  objects,  as  clothing,  dentists'  instruments,  pipes,  dishes,  drinking 
vessels,  etc.,  in  fact  anything  and  everything  which  may  have  been  in 
contact  with  a  syphilitic.  The  primary  lesions  of  the  patients  are  very 
infectious. 


COMMUNICABLE   DISEASES.  2 17 

The  disease  is  readily  preventable.  All  that  is  necessary  is  to  keep  away 
from  the  carriers  of  the  infection.  Syphilitics  should  be  isolated  until 
cured.  The  disease  is  very  readily  kept  under  control  by  the  proper  reme- 
dial agents,  but  persistency  in  the  use  of  medicines  is  necessary  ^ro  effect  a 
cure.  Ehrlich's  606  (Salvarsan),  a  new  remedy  is  considered  in  the  nature 
of  a  specific,  given  in  hypodermic,  intramuscular  or  intravenous  injections. 

M.  Gonorrhea. — This  is  also  a  filth  disease.  The  primary  cause  is  the 
non-sporogenous  Micrococcus  (Diplococcus)  gonorrhea.  It  is  not  infectious 
but  exceedingly  contagious  to  mucous  membranes.  As  Ophthalmia  neonatorum 
(opththalmia  of  the  new-born)  it  is  a  very  fruitful  cause  of  blindness.  The 


FIG.  85. — Gonococcus  and  pus  cells  from  the  urethral  discharges  of  acute  gonorrhea 
The  organism  is  readily  demonstrated  by  the  usual  staining  methods,  using  methylene 
blue  or  Gram's  method.  The  Gonococcus  is  cultured  with  some  difficulty  (use  blood 
serum-agar  in  incubator  at  37°  C.)-  There  are  several  other  cocci  resembling  the  Gono- 
coccus in  form,  but  these  differ  in  that  they  can  be  cultured  in  ordinary  media  at  the  room 
temperature.  (Williams.} 

suppurative  discharges  from  patients  are  highly  contagious.  The  contagion 
is  carried  by  patients  and  by  the  articles  touched  or  handled  by  them.  The 
disease  is  difficult  to  eradicate  from  the  system.  It  is  not  so  frequently 
localized  in  urethra  and  vagina  as  is  generally  supposed,  but  it  may  travel 
to  the  bladder,  kidneys,  joints,  etc.,  and  it  may  be  general  upon  nearly  all 
mucous  membranes  of  the  body.  It  is  very  apt  to  become  chronic,  giving 
rise  to  very  serious  after  effects.  Syphilis  and  gonorrhea  have  the  following 
in  common. 

i.  Both  are  highly  contagious  by  direct  contact,  but  particularly  so  to 
mucous  membranes.  They  are  in  no  sense  infectious  and  are  epidemic 
or  general  only  in  proportion  to  the  number  of  contact  inoculations.  The 


2l8  PHARMACEUTICAL   BACTERIOLOGY. 

chief  carriers  and  disseminators  of  the  contagions  are  the  women  in  public 
houses  and  the  male  frequenters  of  such  houses.  Lack  of  personal  cleanli- 
ness is  a  very  fruitful  source  of  spreading  the  infection. 

2.  The  innocent  (infants,  children  and  adults)  are  occasionally  infected 
through  contact  with  those  afflicted  with  the  diseases,  as  in  shaking  hands, 
kissing,  contact  with  clothing  and  other  articles  used  by  those  already  in- 
fected.    Physicians,  dentists,  and  nurses  may  become  accidentally  infected. 
Physicians  and  dentists  may  inoculate  patients  accidentally,  through  the 
use  of  improperly  disinfected  instruments ;  this  is,  however,  quite  rare.     Con- 
taminated drinking  vessels,  spoons,  forks,  etc.,  may  convey  the  infection. 

3.  In  both  diseases  the  primary  causes  are  readily  destroyed  by  the  use 
of  disinfectants.     With  absolute  cleanliness  the  diseases  could  not  exist. 
In  brief,  the  two  diseases  could  not  exist  if  moral  and  physical  cleanliness 
prevailed. 

4.  Both  diseases  are  difficult  to  cure  as  already  stated.     Both  are  and 
do  become  general  or  systemic  in  character,  and  are  not  local  as  is  generally 
supposed.     Those  suffering  from  these   diseases  should   be  isolated  and 
should  never  be  allowed  to  come  in  close  contact  with  the  innocent. 

5.  Physicians,  pharmacists  and  nurses  should  act  as  public  agents  in 
giving  information  regarding  the  transmissibility  of,  and  the  difficulty  of 
curing  syphilis  and  gonorrhea  and  pointing  to  clandestine  prostitution  as 
the  most  active  source  of  the  contagion.     It  should  be  made  a  criminal 
offense  for  a  syphilitic  to  convey  the  contagion  to  an  innocent  person.     In 
the  army  and  navy  the  men  receive  careful  instruction  as  to  preventive 
measures.     This  was  found  necessary  as  the  prevalence  of  these  diseases 
incapacitated  a  large  percentage  of  the  men  from  active  duty. 

In  the  treatment  and  cure  of  syphilis  mercurial  and  arsenical  preparations 
and  the  iodides  play  a  very  important  part.  In  the  treatment  of  gonorrhea, 
disinfectants,  especially  silver  nitrate  and  protargol,  play  a  very  important 
part.  The  antigonorrheic  bacterin  has  been  used  with  some  success  as  a 
prophylactic  and  as  a  cure  in  chronic  cases.  Only  competent  physicians  can 
treat  these  diseases  properly.  All  advertised  and  patented  "quick  cure" 
remedies  are  fakes. 

Recently  Ehrlich  and  Hata  have  discovered  what  appears  to  be  a  specific 
in  the  treatment  of  syphilis,  namely,  intramuscular  and  intravenous  injec- 
tions of  dioxydia-amidoarsenobenzol  (Salvarsan,  or  "No.  606").  The 
tests  thus  far  made  have  yielded  astonishing  results.  Many  of  the  most 
severe  forms  of  the  disease  have  been  promptly  cured  by  a  single  dose  of 
this  remedy. 

The  Wassermann  or  Wassermann-Noguchi  test  for  syphilis  is  now 
generally  applied  to  determine  whether  or  not  the  Spirochaeta  is  in  the  system. 
The  reaction  is  due  to  certain  bodies  in  the  blood  serum  of  syphilitic  persons 


COMMUNICABLE   DISEASES.  219 

that  display  a  marked  affinity  for  lipoids  and  in  particular,  lecithin.  Many 
workers  now  use,  as  antigen,  an  emulsion  of  lecithin  or  guinea-pig  heart, 
in  place  of  the  watery  emulsion  of  the  liver  obtained  from  a  syphilitic  fetus 
as  described  by  the  originators  of  the  reaction;  the  advantages  being  that 
lecithin  and  guinea-pig's  heart  are  always  on  hand  and  alcoholic  extracts  are 
more  stable  than  watery  extracts. 

The  following  is  an  outline  of  the  method  of  procedure  as  given  by 
George  Gillman  of  San  Francisco. 

i.  Antigen  (a)  (original  Wassermann);  the  liver  of  a  syphilitic  fetus  is 
cut  into  very  small  pieces  and  an  emulsion  made  of  it  by  shaking  with  normal 
salt  solution  (0.85  per  cent.)  in  the  proportion  of  one  (i)  part  of  the  liver  to 
live  (5)  parts  of  the  salt  solution.  After  the  shaking  is  completed,  the 
supernatant  liquid  is  removed  and  clarified  by  centrifugalization,  after 
which  the  clear  liquid  is  pipetted  off,  one-half  of  i  per  cent,  of  phenol 
added  and  stored  on  ice  until  wanted  for  use. 

(b)  If  lecithin  is  to  be  used  as  the  antigen,  it  is  prepared  as  follows:  Make 
up  a  solution  of  pure  lecithin  in  alcohol ;  of  this  alcoholic  solution,  a  quantity 
equal  to  o.i  gm.  of  lecithin,  is  added  to  100  c.c.  of  normal  salt  solution.     This 
is  also  stored  on  ice. 

(c)  Guinea-pig  heart   extract  is  prepared  as  follows:     The   heart  is 
rubbed  up  very  fine  in  a  mortar  (containing  ground  glass)  with  absolute 
alcohol  in  the  proportion  of  one  (i)  gram  of  the  heart  to  25  c.c.  of  absolute 
alcohol.     It  is  then  heated  to  60°  C.  for  an  hour,  filtered  through  filter-paper 
and  kept  in  the  refrigerator  ready  for  use. 

As  the  strength  of  the  antigen  will  vary  in  different  preparations,  it 
must  be  standardized  before  being  used.  It  should  be  of  such  strength 
that  the  quantity  used  will  not  hemolyze  i.o  c.c.  of  a  5  per  cent,  suspension 
of  washed  lamb's  blood-corpuscles  in  the  presence  of  0.2  c.c.  of  a  known 
positive  serum,  o.i  c.c.  of  complement,  and  2  minimal  units  of  the  hemolytic 
serum.  The  unit  is  determined  as  follows :  A  series  of  test-tubes  are  prepared 
each  containing  the  same  quantities  of  the  reagents  mentioned  above  and 
varying  amounts  of  the  antigen.  The  usual  technic  is  followed  and  the 
unit  determined  by  the  quantity  of  antigen  that  inhibited  hemolysis.  After 
this  determination  the  same  antigen  must  be  tested  with  a  known  negative 
serum  used  in  place  of  the  positive  serum  and  using  double  the  unit  of  antigen. 
This  double  unit  should  not  inhibit  hemolysis  of  the  blood  cells.  The  unit 
being  determined,  the  antigen  is  so  diluted  that  i.o  c.c.  will  contain 
the  unit. 

2.  Antibody. — The  blood  serum  or  cerebrospinal  fluid  of  the  syphilitic 
person.  A  sufficient  quantity  of  the  patient's  blood  is  collected  from  the 
lobe  of  the  ear  or  finger  tip,  in  any  sterile  vial  (best  in  a  Wright's  capsule), 
aseptic  precautions,  of  course,  being  observed.  The  blood  is  then 


22O  PHARMACEUTICAL   BACTERIOLOGY. 

centrifugalized  and  the  serum  used.     The  spinal  fluid  is  obtained  in  the 
usual  manner  by  lumbar  puncture. 

3.  Complement. — The  normal  blood  serum  of  a  guinea-pig.     The  blood 
from  one  guinea-pig  is  required,  thus  making  it  necessary  to  sacrifice  one 
animal  for  each  test.     The  blood  must  be  used  fresh,  as  the  serum  loses  its 
complementing  value  if  kept  over  twenty-four  hours.     The  blood  is  defibri- 
nated,  centrifugalized,  and  the  serum  used.     If  stored,  it  should  be  frozen. 

4.  Hemolytic  Serum. — The  blood  serum  of  a  rabbit  that  has  been  injected 
with  washed  lamb's  blood-corpuscles.     The  rabbit  is  immunized  as  follows: 
The  lamb's  blood  is  first  obtained,  best  by  cutting  its  ear  and  allowing  10  c.c. 
of  blood  to  run  into  30  c.c.  of  a  i  per  cent,  sodium  citrate  solution  in  normal 
salt  solution.     (This  will  prevent  the  blood  from  clotting.)     It  is  then 
centrifugalized,  the  supernatant  fluid  pipetted  off,  and  the  blood-corpuscles 
washed  with  normal  salt  solution  by  repeated  centrifugalization  and  dejec- 
tion of  the  supernatant  fluid.    Five  c.c.  of  the  washed  blood-corpuscles  are 
injected  into  the  rabbit  five  (5)  or  six  (6)  times  at  repeated  intervals  of  five  (5) 
days.     On  about  the  tenth  day  after  the  last  injection,  blood  is  taken  from 
the  rabbit,  centrifugalized,  and  the  serum  used.     Before  using  this  serum, 
it  is  necessary  to  test  its  power  after  being  inactivated  (heated  for  three- 
quarters  of  an  hour  at  56°  C.  to  destroy  complement).     The  test  is  made 
to  determine  the  minimum  quantity  of  the  serum  that  will  hemolyze  i  c.c. 
of  the  5  per  cent,  suspension  of  lamb's  blood-corpuscles,  with  o.i  c.c.  of 
complement  (normal  guinea-pig  serum).     Various  quantities  of  the  serum 
to  be  tested  are  put  in  a  series  of  test-tubes  with  i  c.c.  of  the  suspension  of 
lamb's  blood  corpuscles  and  o.i  c.c.  of  the  complement  in  each  tube.     The 
tubes  are  put  in  the  incubator  at  37°  C.,  for  an  hour  and  then  examined  to 
determine  the  smallest  quantity  of  serum  that  produced  hemolysis.     (The 
proper  quantity  is  usually  i  c.c.  of  a  i  in  2000  dilution,  in  normal  salt  solution. 
The  quantity  necessary  for  the  reaction  is  two  minimal  units,  thus  i  c.c.  of  a 
1000  dilution  is  used  for  the  reaction.)     The  dilution  used  should  never  be 
lower  than  i  :iooo.     If  it  happens  to  be  lower  it  will  be  necessary  to  give  the 
rabbit  a  few  more  injections  of  blood-corpuscles,  before  using  its  serum. 

5.  Lamb's  Blood  Corpuscles. — Five  c.c.  of  defibrinated  lamb's  blood  are 
collected  and  washed  with  normal  salt  solution  in  the  same  way  as  the  rab- 
bit's blood.  Then  a  5  per  cent,  suspension  in  normal  salt  solution  is  made. 

The  antigen,  the  patient's  serum  and  the  hemolytic  serum  must  be  in- 
activated (to  destroy  complement)  before  using,  by  heating  them  for  three- 
quarters  of  an  hour  at  56°  C.  The  two  sera  should  be  inactivated  as  soon 
as  made. 

The  antigen,  antibody  (patient's  serum)  complement,  and  hemolytic 
serum  should  each  be  so  diluted  with  normal  salt  solution  that  i  c.c.  of  the 
dilution  will  contain  the  necessary  quantities  needed  for  the  reaction. 


COMMUNICABLE   DISEASES.  221 

Technic  for  Performing  the  Reaction. — Into  a  test-tube  place  0.2  c.c. 
of  the  antigen,  0.2  c.c.  of  the  patient's  serum  (antibody),  and  o.i  c.c. 
of  the  complement.  Incubate  at  37°  C.  for  three-quarters  of  an  hour  and 
then  add  i.o  c.c.  of  the  solution  of  hemolytic  serum,  containing  two  minimal 
doses  and  i.o  c.c.  of  the  5  per  cent,  suspension  of  lamb's  blood-corpuscles. 
Incubate  the  whole  for  two  hours,  place  in  the  refrigerator  over  night,  and 
then  note  if  hemolysis  has  occurred.  If  the  antibody  of  syphilis  is  present  in 
the  suspected  blood  serum,  hemolysis  will  not  occur  because  the  complement 
is  "fixed"  to  the  immune  body  by  the  aid  of  the  antigen  and  the  reaction  is 
positive.  Should  the  suspected  blood  serum  not  contain  the  specific  antibody, 
hemolysis  will  occur  because  there  is  no  immune  body  to  "fix"  the  com- 
plement, therefore  causing  the  hemolytic  amboceptor  (hemolytic  serum), 
by  the  aid  of  the  red  corpuscles,  to  fix  the  complement,  producing  hemolysis 
and  the  reaction  is  then  negative. 

The  substances  employed  are  subject  to  many  external  influences,  and 
it  is,  therefore,  necessary  to  control  their  action.  The  controls  made  are 
necessary  in  order  to  demonstrate  that  none  of  the  employed  substances 
alone  "fix"  the  complement,  and  that  the  occurrence  of  either  a  positive  or 
a  negative  reaction,  when  testing  a  suspected  serum,  is  due  to  and  dependent 
upon  the  fixation  or  non-fixation  of  the  complement  by  means  of  the  immune 
body. 

The  quantity  of  antigen  used  for  the  reaction  may  have  to  be  either 
increased  or  decreased.  The  controls  will  indicate  when  a  change  is  required 
and  the  proper  quantity  necessary  is  determined  by  the  method  given  under 
the  preparation  of  the  antigen. 

There  are  many  other  communicable  diseases  as  measles,  mumps, 
scarlet  fever,  and  whooping  cough,  besides  the  diseases  due  to  the  attacks  of 
higher  parasites,  as  itch,  trichinosis,  tapeworm,  roundworm,  liver  flukes, 
hookworm,  etc.,  which  we  will,  however,  not  discuss  more  fully.  The  sug- 
gestions given  under  the  diseases  described  will  also  apply,  in  a  measure, 
to  other  communicable  diseases.  Summed  up  briefly,  preventive  medicine, 
direct  and  indirect,  consists  of  giving  heed  to  the  following. 

1.  Living  in  accord  with  the  most  approved  methods  of  hygiene.     This 
is  direct  preventive  medicine. 

2.  Treating  disease  in  accord  with  the  most  approved  modern  methods. 
This  is  indirect  preventive  medicine  because  it  protects  the  well  against 
infection  from  the  sick. 

The  following  table  of  communicable  diseases  giving  the  average  period 
of  incubation  (also  known  as  latent  period),  the  primary  cause,  nature  of 
communicability  and  principle  carriers  or  sources  of  infection,  will  be 
found  useful. 


222 


PHARMACEUTICAL   BACTERIOLOGY. 


Incubation 

Name  of  disease.        period,     I      Primary  cause, 
days. 

I 


Nature  of 
communicability. 


Carriers  or  sources 
of  infection. 


Anthrax    or  wool 


sorter  s  disease. 

tagious. 

Bubonic  plague    . 

4-6 

Bacillus  pestis 

Rats  mice  fleas  filth 

Asiatic  cholera.  .  . 

2-4 

Bacillus  cholerae.  .  . 

Infectious    and    con- 
tagious. 

Flies,    polluted    water 
and  food. 

Diphtheria 

Bacillus  diphtheria? 

tagious. 

sick. 

Erysipelas  

4-6 

Streptococcus  pyo- 

Very    contagious     to 

Dirt       perhaps     flies 

genes. 

wounds. 

mosquitos. 

Influenza,  Grippe  j          1-4        I  Bacillus  influenzse. 


Infectious,   not    con- 
tagious. 


Air,   and  exposed  ob- 
jects. 


Glanders  

3-5 

Bacillus  mallei  

Contagious  and  infec- 
tious. 

Horse  and    horse-like 
animals. 

Gonorrhea 

Micrococcus  gonor- 

rheaj. 

infectious. 

jects. 

Mumps     

10-16 

Unknown  

Very  infectious      .  .  . 

Air  and  the  sick 

Malaria  

6—  10 

Plasmodium  mal- 

Infectious,    not    con- 

Mosquitos (Anophe- 

ariae. 

tagious. 

les). 

Relapsing  fever.  . 

5-6 

Spirochaeta  Ober- 
meieri. 

Infectious  

Insects,   as   bed  bugs, 
etc. 

Measles.  

8-9 

Unknown  

Very  contagious,  also 
infectious. 

Exposed  objects. 

Hydrophobia 

Contagious  to  wounds 

other  canines. 

Rubeola,  rubella.  . 

18 

Unknown  

Infectious    and    con- 
tagious. 

Exposed  objects. 

Scarlatina  

2-5 

Unknown,  perhaps 
Protozoa. 

Infectious    and    con- 
tagious. 

Exposed  objects. 

Small-pox  

12 

Unknown  

Infectious    and    very 
contagious. 

Exposed  objects. 

Syphilis  

14-30 

Spirochaeta  pallida. 

Very   contagious,  es- 
pecially to  lesions. 

Exposed  objects. 
A  filth  disease. 

Tetanus,  lock-jaw 

2-3 

Bacillus  tetani  

Contagious  to  lesions 
only. 

Dirt,   infected  objects 
of  all  kinds. 

Typhoid  fever..  .  .  \          14  Bacillus  typhosus.  . 


Infectious    and    con- 
tagious. 


Polluted     water     and 
food.     Flies. 


COMMUNICABLE   DISEASES. 


223 


Name  of  disease. 


Incubation 

period, 

days. 


Primary  cause. 


Nature  of 
communicability. 


Carriers  or  sources 
of  infection. 


Vaccinia. . . 


3-6 


Unknown Contagious  by  inocu- 
lation only. 


Cow     virus,      human 
vaccinia. 


Varicella,    chick- 
en-pox. 


14-15          Unknown Contagious Those  affected. 


Whooping  cough.. 


Unknown Very  infectious 


Exposure      to      those 
affected. 


Yellow  fever.  .  .  . 

3-4 

Unknown,  plasmo- 
'     dium? 

Infectious,    not    con- 
tagious. 

Mosquitos  (Stegom- 
yia.) 

Leprosy 

Weeks 

Bacillus  leprae 

Infectious    and    con- 

The patients 

1     months, 
years. 

tagious. 

Tuberculosis.  .  .  . 

.       Weeks 
and 
longer. 

Bacillus      tubercu- 
losis. 

Infectious  

Sputum,  milk  from 
tubercular  cows. 

Dengue 


Protozoa  ? . .  .  .    Infectious . 


Mosquito    (culex  fati- 
gans) . 


'Pneumonia 


Micrococcus    pneu-     Infectious, 
moniae   (Diplococ- 

cus). 


Carried  by  persons. 


Dysentery  (bacil- 
lary) 


Amoeba  dysenteriae.    Infectious Polluted  water  supply. 


Malta  fever.  .  . 


Beri-beri.  . 


6-10 


Micrococcus     meli-     Infectious 

tensis. 


Goats'  milk,  stings  of 
insects. 


Months.       Micrococcus? Infectious A  tropical  disease. 


Pellagra. 


Aspergillus  species..    Neither  infectious  nor 
contagious. 


Mouldy     foods,     corn 
especially. 


In  some  diseases  the  mortality  rate  is  very  high,  as  in  yellow  fever, 
beri-beri,  tetanus,  cholera,  plague  and  leprosy.  In  others  it  is  low,  as  in 
syphilis,  gonorrhea,  malaria,  whooping  cough,  mumps  and  varicella.  In  cer- 
tain diseases  the  prognosis  is  rather  uncertain,  the  mortality  rate  being  high 
at  times  and  again  low,  as  in  scarlatina,  small-pox,  measles  and  grippe.  Some 
diseases  run  a  somewhat  variably  rapid  course  as  pneumonia,  diphtheria, 
spinal  meningitis,  bubonic  plague  and  Asiatic  cholera,  ending  either  in  death 
or  recovery.  Other  diseases,  as  scarlet  fever,  measles  and  diphtheria  may 
have  after-effects  or  sequelae  which  often  assume  a  chronic  course  and  may 
finally  result  in  death.  Certain  diseases  run  a  regular  course  which  varies 
but  little  as  to  the  sequence  of  symptoms  and  duration,  as  typhoid  fever 
(five  weeks).  Others  run  a  variably  chronic  course,  ending  either  in  death 


224  PHARMACEUTICAL   BACTERIOLOGY. 

or  recovery,  as  pellagra  and  malaria.  Some  diseases  are  very  persistent, 
difficult  to  eradicate  from  the  system,  showing  certain  effects  even  to  the 
third  and  fourth  generations,  as  tuberculosis  and  syphilis.  Malaria  leaves 
certain  after-effects,  as  enlarged  spleen  ("ague  cake"),  which  may  persist 
through  life. 

Savage  races  are  peculiarly  susceptible  to  certain  diseases,  as  tuberculosis, 
small-pox,  gonorrhea  and  syphilis  and  peculiarly  enough  these  diseases  did 
not  originate  with  primitive  peoples,  but  with  advanced  civilization,  though 
of  great  antiquity. 


CHAPTER  XIV. 

A  BACTERIOLOGICAL  AND  MICROSCOPICAL  LABORATORY  FOR 

THE  PHARMACIST. 

The  pharmacist  imbued  with  the  proper  appreciation  of  his  responsibility 
in  the  community  should  equip  a  laboratory  in  which  to  do  the  necessary 
and  desirable  work  in  bacteriology  and  microscopy.  The  pharmacist, 
more  than  any  other  professional  man,  has  or  should  have  the  opportunity 
to  do  laboratory  work.  The  significance  of  bacteriology  in  pharmacy  has 
been  sufficiently  set  forth  in  the  pages  of  this  book.  The  details  of  the 
microscopical  work  proper  belongs  to  the  field  of  botany  and  pharmacognosy 
and  cannot  be  fully  explained  here.  Every  graduate  from  a  reputable 
college  of  pharmacy  is  or  should  be  qualified  to  use  the  microscope  in  the 
examination  of  vegetable  drugs  (crude  and  powdered),  of  compound  powders, 
dusting  powders,  insect  powders,  starches,  meals,  flours,  including  foods  of 
vegetable  origin  as  baby  foods,  breakfast  foods,  etc. 

The  following  suggestions  will  no  doubt  be  useful,  though  it  is  not  in- 
tended to  imply  that  they  must  be  carried  out  strictly.  The  laboratory  space, 
location,  equipment,  etc.,  can  be  made  to  suit  individual  requirements. 
The  microscopical  work  should  preferably  be  done  in  a  separate  room,  not 
in  the  bacteriological  laboratory,  though  this  is  not  absolutely  essential. 

A.  Location  of  Laboratory. — It  may  be  in  a  separate  building,  as  the 
home,  but  as  a  rule  a  corner  room  in  the  pharmacy  is  best  suited  for  the 
purpose.  This  room  may  be  in  the  basement,  or  on  the  first,  second  or  other 
floor.  Do  not  select  a  room  with  a  through  passage  for  obvious  reasons. 
It  may  adjoin  a  chemical  or  pharmaceutical  laboratory,  though  it  should  not 
be  a  part  of  such  laboratories.  Chemicals  and  chemical  fumes  interfere 
with  bacteriological  and  microscopical  work.  It  should  have  one  door  and 
two  or  more  windows.  There  must  be  good  light  and  the  environment 
should  be  favorable  for  bacteriological  work,  for  which  reason  a  room  in  the 
basement  is  not,  as  a  rule,  desirable. 

The  walls  and  ceiling  of  this  room  should  be  absolutely  plain  and  well 
protected  with  white  enamel  paint.  The  floor  may  be  cement,  slate,  or  hard 
wood  well  oiled  with  boiled  linseed  oil,  or  it  may  be  painted,  or  covered 
15  225 


226  PHARMACEUTICAL   BACTERIOLOGY. 

with  linoleum.  The  entire  room  (walls,  ceiling,  floor)  should  be  washed, 
scrubbed  and  disinfected  from  time  to  time.  That  is,  it  should  be  kept 
bacteriologically  clean. 

B.  Furnishings. — All  windows  exposed  to  direct  sunlight  should  have 
white  translucent  roller  shades.  The  laboratory  should  be  well  supplied 
with  gas;  water,  both  hot  and  cold,  if  possible;  and  means  for  lighting 
(gas,  electricity,  acetylene).  There  should  be  just  enough  furniture  and 
shelving,  no  more.  One  table  with  slate  top  or  lined  with  linoleum;  one 
stool,  shelves  for  samples,  apparatus  and  reagents.  A  case  for  chemicals, 
cotton,  culture  media,  etc.  A  case,  with  lock  and  key,  for  samples  to  be 
examined.  The  plumbing  must  be  of  the  best  and  the  fixtures  must  be  of 
safe  construction.  The  sink  should  be  large  and  deep  and  should  be  lined 
with  porcelain  and  supplied  with  an  ample  drain  board.  A  hood  or  ventila- 
tor should  be  provided  to  carry  off  steam  vapors.  Near  the  table  for  micro- 
scopical work  should  be  a  shelf  or  case  for  the  following  works  of  reference: 
U.  S.  Pharmacopoeia,  National  Formulary;  National  Dispensatory;  Sayre's 
Organic  Materia  Medica  and  Pharmacognosy;  Culbreth's  Materia  Media 
and  Pharmacology;  Kraemer's  Botany  and  Pharmacognosy;  Schneider's 
Powdered  Vegetable  Drugs ;  Winton's  (Moeller's)  Microscopy  of  Vegetable 
Foods;  Brundage's  Manual  of  Toxicology;  Holland's  Urine,  Common 
Poisons  and  Milk;  Muir  and  Ritchie's  Manual  of  Bacteriology;  Official 
Methods  of  the  American  Association  of  Agricultural  Chemists.  There  are 
many  other  desirable  reference  works,  but  the  above  will  serve  as  a  nucleus 
to  which  additions  can  be  made  from  time  to  time.  Only  the  latest  edi- 
tions should  be  purchased. 

C.  Apparatus. — There  will  be  required: 

a.  A  good  simple  lens. 

b.  Compound  miscroscope.     (Leitz,  Zeiss  or  Bausch  and  Lomb). 
Ocular  with  micrometer  scale. 

Oculars,  Nos.  2  and  3. 

Objectives,  Nos.  3,  5,  7  and  1/12  oil  immersion. 

c.  Slides  and  covers. 

d.  Section  knife  or  razor,  and  strop. 

e.  Polarizer,  for  the  study  of  starches,  crystals,  etc.     Should  be  conven- 
ient to  use.     This  is  very  important.     The  selenite  plates  which  are  usually 
supplied  with  the  polarizer  are  useful. 

f .  Thoma-Zeiss  hemacytometer  with  Turck  ruling,  for  counting  bacteria, 
spores  and  yeast  cells  in  vinegar,  jams,  jellies  and  other  like  substances. 

g.  Accurately  ruled  metal  or  hard  rubber  millimeter  ruler  for  measuring 
seeds  in  fruit  products,  etc. 

h.  One  Arnold  steam  sterilizer  (copper).     A  vegetable  steam  cooker 
will  serve. 


A   BACTERIOLOGICAL  AND   MICROSCOPICAL   LABORATORY.  227 

i.  One  hot  air  sterilizer.  The  ordinary  double  walled  baking  ovens 
which  may  be  secured  from  any  hardware  dealer,  will  serve  the  purpose. 
Cut  in  a  small  opening  at  top  for  the  thermometer. 

j.  One  rice  cooker  in  which  to  prepare  culture  media,  etc. 

k.  One  small  incubator  with  Reichert  thermo-regulator. 

1.  Centrifuge  (electric  or  water  motor). 

In  addition  to  the  above  there  will  be  required  the  necessary  chemicals, 
reagents,  etc.,  good  quality  commerical  cotton  for  plugging  test-tubes, 
medium  size  Petri  dishes,  flasks  (1/2  liter  and  i  liter),  several  evaporating 
dishes,  one  or  two  moist  chambers,  a  quantity  of  medium  size  test-tubes, 
slide  boxes,  test-tube  brushes,  dissecting  needles,  scalpels,  labels,  pencils, 
etc.  Get  the  necessary  things  only.  There  must  be  a  liberal  supply  of 
clean  towels.  No  one  but  the  analyst  and  his  assistants  should  have  access 
to  the  laboratory.  On  entering,  the  analyst  should  remove  coat  and  hat 
and  put  on  a  white  clean  linen  apron  and  coat,  such  as  are  worn  by  soda 
fountain  dispensers.  This  white  suit  should  remain  in  the  laboratory  and 
should  be  changed  for  a  clean  one  as  often  as  may  be  necessary. 

Special  equipment  and  apparatus  may  be  indicated  as  the  work  pro- 
gresses. For  instance,  it  may  prove  desirable  to  have  an  incubator  for 
opsonic  work,  for  the  use  of  physicians,  used  either  by  the  physicians 
or  by  the  pharmacist.  A  water  filtering  equipment  may  be  installed,  like- 
wise a  water  still.  An  autoclave  may  prove  desirable.  There  are  matters 
which  must  be  left  to  the  individual  pharmacist.  The  following  is  an 
outline  of  such  work  as  the  pharmacist  may  do  in  the  microscopical  and 
bacteriological  laboratory. 

D.  Micro-analytical  Work. — The  skilled  microscopist  should  be  ready 
to  determine  the  quality  and  purity  of  the  following  substances: 

a.  Vegetable  drugs,  crude  and  powdered. 

b.  Spices  and  condiments,   whole,   ground   and   powdered.     Prepared 
spices  and  condiments. 

c.  Coffee,  tea,  cocoa,  chocolate,  confections,  candies. 

d.  Tobacco,  including  smoking  tobacco,  cigars,  cigarettes,  snuff. 

e.  Compound  powders,  pharmacopceial  and  others. 

f.  Tablets,  pills,  simple  powders. 

g.  Meats ;  raw,  cooked,  canned,  sausage  meats,  mince  meats,  etc. 

h.  Dairying  products  as  milk,  cream,  cheese,  butter,  ice  creams,  cream 
fillers. 

i.  Cosmetics,  dusting  powders,  insect  powders. 

j.  Cattle  and  poultry  powders. 

k.  Starches,  dextrins,  sausage  meat  binders  (starchy). 

1.  Vegetable  foods;  as  jams,  jellies,  fresh,  pickled,  cooked,  canned  and 
preserved. 


228  PHARMACEUTICAL    BACTERIOLOGY. 

m.  Flours  and  meals. 

n.  Breakfast  foods,  baby  food,  invalid  foods. 

0.  Breads,  cakes,  pies,  crackers,  etc. 
p.  Catsups,  tomato  pastes,  etc. 

'q.  Macaroni,  spaghetti,  noodles,  etc. 

r.  Nuts,  and  nut-like  fruits. 

s.  Cloth  material,  textile  fabrics  generally,  cordage,  papers,  etc. 

It  is  assumed  that  the  pharmacist  has  had  the  necessary  training  to  under- 
take the  microscopical  examination  of  the  substances  above  classified,  with 
the  aid  of  such  standard  works  of  reference  as  may  be  required.  The 
micro-analyses  should  also  include: 

1.  Gross  and  net  weight  determination  of  all  samples  that  require  it, 
for  which  purpose  an  accurate  balance  is  necessary. 

2.  Moisture  determinations  of  such  substances  as  may  require  it.     There 
should  be  no  difficulty  in  constructing  the  necessary  apparatus  for  making 
moisture  determinations. 

3.  Ash  determinations  of  substances  which  require  it.     This  calls  for 
a  special  equipment  including  a  platinum  dish,  ignition  furnace  with  burners, 
etc. 

4.  Use  of  special  tests,  as  sublimation  tests  for  benzoic  acid  and  salicylic 
acid,  the  hand  wheat  gluten  test,  Bamihl  gluten  test,  Grahe's  cinchona  test, 
color  reaction  tests  for  boric  acid,  salicylic  acid,  morphine,  and  opium; 
tests  for  phytosterol  and  cholesterol  crystals,  etc.,  etc.    These  and  other  tests 
are  explained  in  the  several  reference  works  cited  above.     In  the  examina- 
tion of  liquids  or  semi-liquids  as  wines,  beers,  cider,  vinegar,  milk,  cream, 
sewage,  extracts,-  tinctures,  etc.,  a  centrifuge  is  desirable. 

E.  Bacteriological  Work. — :The  pharmacist  should  be  prepared  to  do  the 
following  work  in  the  bacteriological  laboratory. 

a.  Prepare  culture  media  for  use  of  physicians,  as  may  be  required. 

b.  Prepare  sterile  throat  swabs  for  the  use  of  physicians. 

c.  Prepare  stains  and  do  staining  for  physicians,  as  may  be  required. 

d.  Make  bacteriological  determinations  of  milk,  jams,  jellies,  impure 
drinking  water,  vinegar,  wine,  sera,  vaccines,  antitoxins,  contaminated  foods 
and  drinks,  sewage,  etc. 

e.  Sterilize  pharmaceuticals,  surgical  supplies,  etc. 

f .  Assist  the  physician  in  opsonic  work,  as  may  be  arranged  or  agreed 
upon. 

g.  Do  bacterial  culture  incubation  work  for  the  physician,  make  sub- 
cultures, Wassermann  reaction  for  syphilis,  etc. 

h.  Filter  and  sterilize  drinking  water  to  be  supplied  to  customers. 
The  above  outline  is  intended  as  a  suggestion  only.     Experience  and 
opportunity  will  determine  what  can  actually  be  done. 


A   BACTERIOLOGICAL  AND   MICROSCOPICAL   LABORATORY.  22Q 

The  following  is  a  diagram  of  a  bacteriological  and  microscopical  labor- 
atory: 


0 


FIG.  86. — Plan  of  bacteriological  and  microscopical  laboratory,  using  corner  room 
in  the  pharmacy.  (Scale  4  feet  to  the  inch.)  A,  shelves  on  three  sides  of  the  room. 
B,  work  table.  C,  cases  and  shelves  for  reagents,  chemicals,  glassware,  etc.  D,  case  for 
reference  books.  E,  sink.  F,  drain  board,  i,  Arnold  steam  sterilizer;  2,  hot  water 
filter;  3,  hot  air  sterilizer;  4,  rice  cooker;  5,  opsonic  incubator;  6,  incubator;  7,  compound 
microscope;  8,  stool;  9,  hat  and  coat  hooks. 


INDEX. 


Acharion,  143 
Acid  stains,  70 
Acetanilid,  169 
Actinomyces,  146 
Aerobioscope,  61,  62 
Aetius,  6 

African  tick  fever,  157 
Agar  medium,  46 
Agglutination,  122 
Agitation,  162 
Agricultural  bacteria,  93 
Air,  bad,  161 

pure,  161 
Albumoses,  94 
Alfalfa,  96 
Alinit,  97,  99 
Alpinus,  6 
Alumn,  185 
Amboceptors,  123 
Ammonia,  94 
Ampuls,  195 
Anapholes,  158 
Anaximander,  8 
Anchovy  pastes,  67 
Aniline  water,  72 
Antagonism,  32 
Antidiphtheric  serum,  129 
Antigens,  122 
Antiplague  serum,  135 
Antipyrin  169 
Antiseptics,  159 

active,  170 

cost  of,  164 

respiratory,  170 

weak,  171 

Antitetanic  serum,  134 
Antitoxin,  116 
Ants,  189 
Aphis,  in,  187 
Arcana  naturae,  8,  9 
Aristol,  168 
Aristotle,  8 
Armour's  extract,  43 
Arnold  sterilizer,  39,  42 
Arrak,  153 
Arsenic,  186 
Arthrospores,  28 
Ascomycetes,  143 
Asiatic  cholera,  14 
Aspergillus,  32,  145 
Audry,  10 
Autoclave,  40 
Azoa,  in 
Azotobacter,  94,  99 


Babes-Ernest  granules,  26 
Bacillary  tablets,  no 
Bacilli,  morphology  of,  24 
Bacillus,  23 

anthracis,  203 
acidi  lactici,  100,  101 
actinobacter,  101 
bulgaricus,  108 
butyricus,  101 
californiensis,  94 
of  cholera,  27 
cholerse,  214 
coli  communis,  44 
colon,  63,  67,  68 
comma,  15 
cyanogenus,  101 
diphtheria,  15,  28,  212 
of  dysentery,  106 
ellenbachiensis,  94 
erodiens,  112 
gasoformans,  112 
gummosus,  101 
of  Koch,  207 
of  lactic  acid,  100,  ic8 
lactis  viscosus,  101 
mallei,  203,  205 
megaterium,  97 
melitensis,  106 
mesentericus,  32 
murisepticus,  in 
•  para- typhoid,  122 
pestis,  135,  214 
pneumonias,  210 
prodigiosus,  32,  141 
pyocyaneus,  32 
subtilis,  32,  94 
tetani,  205 
tuberculosis,  207 
typhimurium,  in 
typhosus,  69,  209 
xylenum,  113 
Bacteremia,  34 
Bacteria,  in  agriculture,  93 
of  air,  6 1 

in  canned  foods,  64 
classification,  21,  22 
counting  of,  66,  67 
distribution,  33 
food  of,  30 
industrial,  93 
intestinal  33,  94 
of  liquids,  62 
in  milk,  33,  100,  103 
in  certified  milk,  103 


23I 


232 


INDEX. 


Bacteria,  morphology  of,  21,  24 

mounting,  91 

mouth,  33 

naming  of,  77 

nitrifying,  94 

in  pharmaceuticals,  2 

phosphorescent,  31 

physiology  of,  21.  28 

range,  33 

root  nodule,  61 

rotting,  31,  94,  112 

size  of,  2 1 

in  soil,  59 

staining,  70 

studying,  76 

in  mineral  waters,  64 
Bacteriaceae,  23 
Bacterial  chart,  78 

counts,  59 

cultures,  52 
Bacterins,  135 
Bacteriological  laboratory,  225,  229 

work,  228 
Bacteriology,  history  of,  5 

medical,  i 

in  pharmacy,  i 
Bacterolysin,  116 
Baker,  Henry,  10,  18 
Basic  stains,  70 
Baskets,  wire,  37 
Bassi,  13 
Bastian,  n 
Bating,  112 
Beds,  contact,  112 
Beef  broth,  45 

extract,  43 

Beer  making,  148,  149 
Beggiatoa,  23 
Begiatoaceae,  23 
Behring,  14,  116 
Berzelius,  10 
Betol,  169 

Bichloride  of  mercury,  167 
Billroth,  13,  14 
Bleaching  wool,  9 
Blissus,  no 
Blood  serum,  40,  44 
Blue,  Rupert,  in 
Blue  vitriol,  168 
Boiling  milk,  104 
Borax,  169 

Bordeaux  mixture,  187 
Boric  acid,  169 
Bottles,  reagent,  36 
Bougie,  filter,  56 
Bouillon,  nutrient,  43 

sugar  free,  44 
Bovine  tuberculosis,  103 
Braconnot,  10 
Brieger,  16 
Broth,  beef,  45 
Buchner,  16 
Burner,  ring,  52 


Butter  fat,  103 
milk,  no 
ripening  of,  100 

Cadaver  disinfection,  175 
Cagniard-Latour,  10 
Calmette,  16 
Campbell,  H.  P.,  3 
Cancer,  212 
Cancer  vaccine,  141 
Canned  fruits,  64 
Capsule  staining,  76 
Carbolic  acid,  166 
Carbon  bisulphide,  186 
Cardan,  8 
Caron,  97 

Catgut,  disinfection,  194 
Cell  receptors,  120 
Cheese,  American,  107 

Bre,  101 

Camembert,  107 

Cheddar,  107 

Edam,  107 

hard,  107 

Limburger,  101 

ripening  of,  100,  107 

Roquefort,  107 

soft,  107 

Swiss,  107 
Chinatown,  7 
Chinch  bug,  no 
Chinese  yeast,  155 
Chlorophyll,  26 
Cholera,  14,  214 
Cider,  hard,  149 

making,  113 
Cilia,  26 
Circulation,  162 
Cladothrix,  23 
Clarification,  of  media,  51 
Cleaning  glassware,  36 

solution,  37 
Cleanliness,  160 
Clostridium,  99 
Clover,  red,  95 

sweet,  96 

white,  98 
Clumping,  122 
Coccaceae,  22 
Cocci,  morphology,  24 
Cohn,  13 

Colbach,  John,  12 
Cold,  30,  31,  162 
Collargol,  119 
Colon  bacillus,  63 

group,  69 
Colonies,  58 

bacterial,  87 
Comma  bacillus,  15 
Communicable  diseases,  202 
Concentrated  antitoxin,  133 
Conn,  H.  W.,  18 
Contact  beds,  112 


INDEX. 


233 


Copeman,  S.  M.,  18 
Copperas,  168 
Copper  sulphate,  168 
Corrosive  sublimate,  167 
Cotton,  for  test  tubes,  37 
Counting  bacteria,  66 
Counting  plates,  88,  90 
Counting  plate  colonies,  87 
Counts,  bacterial,  59 

plate  culture,  67 
Cover-glass  pincers,  53 

mounts,  91 

Cows,  tuberculous,  103 
Cream,  ripening,  101,  107 

sour,  107 
Crenothrix,  23 
Creolin,  166 
Creosote,  169 
Cresol,  166 

Crookshank,  E.  M.,  18 
Crop  rotation,  93 
Cryptococcus,  143 
Culture  media,  38 

preparation  of,  41 

sterilization,  39 
Cultures,  isolation,  57 

notes  on,  55 

test  tube,  45,  54 

swab,  43 
Curing,  cacao,  112 

tobacco,  112 

tea,  112 

Cutaneous  test,  208 
Cycle,  life,  29 

Decay,  cause  of,  10 

Deep  stab  culture,  56 

Definitions,  78 

DeFoe,  Daniel,  18 

Depilation,  112 

Detre  differential  test,  208 

Devaine,  13 

Dhobie's  itch,  146 

Diplococcus  meningitidis,  135 

Diphtheria,  212 
bacillus,  15,  28 

Dirt,  161 

Discomyces,  146 

Diseases,  causes  of,  203,  206 
communicable,  202 
quarantinable,  177 

Disinfectants,  159 
action  of,  164 
chemical,  163 
standardization,  160 

Disinfection,  171,  190 
of  excreta,  172 
in  pharmacy,  190 
public  buildings,  176 
of  sick  room,  172,  173 
of  wounds,  12 

Division,  28,  29 

Dolley,  C.  S.,  18 


Double  staining,  71 
Dourine,  157 
Drenching,  112 
Dulcin,  185 

Earthquakes,  6,  7 
Eccles,  R.  C.,  3 
Echinacese,  123 
Effluvias,  5,  6 
Ehrenberg,  10 
Ehrlich,  Paul,  16,  19,  116 
Eisenberg,  22 
Electricity,  163 
Ellis,  David,  19 
Embalming  fluid,  175 
Emmerich,  16 
Empedocles,  8 
Endomyces.  143 
Endospores,  27 
Entamceba,  156 

coli,  156 
Enzymes,  17 
Erythrasma,  146 
Esmarch  roll  culture,  55 
Europhen,  168 
Eyre,  J.  W.;  19 

Fat,  26 

butter,  103 
Favus,  143 
Fermenlactyl,  no 
Fermentation,  prevention  of,  10 

tubes,  60 
Fertility,  93 

kinetic,  93 

potential,  93 

of  soil,  93 

Fertilizer,  microbic,  96 
Filling  test  tubes,  38 
Filter  bougie,  56 

Kitasato,  56 
Filtering,  49 
Filth,  161 
Filtration,  162 
Fischer,  22 
Fission  fungi,  22 
Flagellae,  24 
Flagellar  staining,  75 
Fliigge,  1 6 
Fluorine,  185 
Food,  mouldy,  148 

preservatives,  181 
Formalin,  166 

disinfection,  174 
Frank,  17 
Fraenkel,  15 
Freezing,  30 
French  gelatin,  48 
Frost,  W.  D.,  19 
Fruits,  canned,  64 
Funnel,  hot  water,  52 

Galen,  6 
Gases,  186 


234 


INDEX. 


Gas  formation,  68 

formula,  68 

regulator,  47 
Gelatin,  French,  48 

medium,  45 
Generation,  de  novo,  8 

spontaneous,  10,  u 
Germicides,  159 
Gilman,  141 
Glanders,  205 
Glass  rods,  52 
Glassware,  cleaning,  36 
Glossary  of  terms,  78 
Glycine  hispida,  153 
Gonorrhea,  217 
Gophers,  186 
Gram's  stain,  72 
Green  manuring,  94 
Gruber-Widal  test,  210 
Guaiacol,  169 
Guinea-pigs,  132 

Habits,  pernicious,  205 
Haffkine's  vaccine.  127 
Hallier,  13 
Hanging  drop,  89 
Haptophore,  120 
Harrocks,  W.  H.,  19 
Hartleb,  97 
Hatch,  J.  L.,  3 
Heat,  162 
Hektoen,  118 
Hemacytometer,  66 
Hemolysin,  116 
Henle,  13 
Hewlett,  R.  T.,  3 
Killer,  13 
Hiltner,  96 
Hindus,  6 
Hinnean,  120 
Hip-joint  disease,  207 
Hippocrates,  5,  6 
Hodges,  7 
Hog  cholera,  68 
Hog  erysipelas,  15 
Homunculus,  8 
Hooke,  Robert,  10,  19 
Hoppe-Seyler,  13 
Horse,  bleeding  of,  129 
Hot-air  sterilizer,  38 
Hot  water  funnel,  52 
Howard,  L.  O.,  19 
Hueppe,  1 6 

Humboldt,  A.  von,  n,  12 
Hydrocyanic  acid,  187 
Hydrogen  dioxide,  169 
Hydrophobia,  140 
Hypergrowth,  98 
Hypernutrition,  98 
Hypodermic  solutions,  199 

Immunity,  15,  114 
Immunization,  124 


Incubation  period,  222 
Incubator,  46 
Index,  opsonic,  118 
Infectionsfaden,  95 
Infections,  mixed,  53 
Infusoria,  10 
Insecticides,  185 
Insecticide  table,  188 
lodoform,  168 
lodol,  169 
Iron  deposits,  31 
Iron  sulphate,  168 
Isolation  cultures,  57 

Jeffer's  counting  plate,  90 
Jelliffe,  Smith  Ely,  3 
Jenner,  12 
Jetter,  16 
Jordan,  E.  O.,  19 

Kefir,  1 08 

grains,  109 

powder,  109 

preparation  of,  109 

seeds,  109 
Kircher,  8,  9 
Kitasato,  15,  116 

filter,  56 
Klebs,  13 
Klein,  E.,  3 
Koch,  Robert,  13,  15 
Koch's  lymph,  16 
Kraemer,  180 
Kraus,  122 
Kuhne,  13 

Laboratory,  bacteriological,  225,  229 

Lactic  acid  bacillus,  100 

Lafar,  F.,  19 

Lacto-bacilline,  no 

Lactone  tablets,  no 

Laking,  116 

Laudable  pus,  13,  14,  117 

Leeuwenhoek,  5,  8 

Leucocytes,  16,  117 

Liebig's  extract,  43 

Life  cycle,  29 

Light,  163 

Lime,  168 

chlorinated,  168 

milk  of,  1 68 

slaked,  186 
Liq.  cres.  comp.,  166 
Lister,  14 
Listerine,  14 
Listerism,  14 

Literature,  bacteriological,  3,  18 
Lobelia,  123 
.Loffier,  15 

Loeffler's  blood  serum,  43 
Losophen,  169 
Lymans,  W.  H.,  4 


INDEX. 


235 


Lymph,  Koch's,  16 
Lysins,  116 

Maggots,  10 

Malaria,  158,  212 

Mai  de  caderas,  157 

Marcellinus,  6 

Marpmann's  vaccine,  127 

Mead,  8 

Meat  preservatives,  184 

putrid,  10 

smoking  of,  185 
Media,  clarification,  51 

culture,  47 

preparation  of,  41,  47 

neutralization,  49 
Medical  bacteriology,  i 
Medicines,  disinfection  of,  192 
Medium,  agar,  46 

gelatine,  45 

Mercuric  chloride,  167 
Mesophile,  30 
Metachromes,  26 
Metchnikoff,  16,  108 
Mez,  K.  C.,  19 
Micro-analytical  work,  227 
Microbic  fertilizer,  96 
Microbiology ,  21 
Micrococci,  22 
Micrococcus,  23 
Micrographia,  10 
Microscopes,  226 
Microscopical  examination,  89 

work,  225 
Microspira,  23 
Migula,  22 
Milk,  bacteria  of,  100 

boiling,  104 

bottling,  1 02 

condensed,  106 

certified,  102 

dry,  1 06 

evaporated,  106 

fat,  103 

freshly  drawn,  101 

full,  103 

half,  103 

klabbered,  108 

of  lime,  1 68 

medium,  44 

pasteurization,  105 

preservation,  104 

ropy,  10 1 

skimmed,  103 
Mineral  waters,  64 
Mites,  9 

Montagu,  Lady,  n 
Mordants,  71 
Moro  test.  208 
Mosaic  law,  9 
Mosquitos,  158 
Motility,  27 
Motion,  rate  of,  27 


Mould,  65 

counter,  65 

culture  of,  147 
Moulds,  142 
Mouldy  food,  148 
Mounts,  bacterial,  91 
Mouratus,  in 
Mouth  bacteria,  33 
Mucor,  142 
Miiller,  10 

Murrill's  gas  regulator,  47 
Muscardine  disease,  13 
Mutualism,  32 
Mycobacteriaceae,  23 
Mycoderma  aceti,  113 

Naphthaline,  169 
Naphthol,  169 
Neufeld,  118 
Neutralization,  49 
Neutral  red  reduction,  68 
Newman,  Geo.,  20 
Nicolaier,  15 
Nitragin,  96 
Nitrogen,  94 
Nobbe,  96 
Noguchi,  218 
Nomenclature,  77 
Normal  solutions,  49 
Nosophen,  169 
Nuclein,  123,  155 
Nucleinic  ac'd,  119 
Nucleoplasm,  24 
Nutrient  bouillon,  43 

Oidium,  107 
Ophthalmo  test,  208 
Opsonic  incubator,  119 

index,  118 
Opsonins,  16,  118 
Opsonogens,  119 
Ovid,  8 

Panum,  14 
Paracelsus,  8 
Paramecium,  30 
Parasite,  30 
Paris  green,  186 
Paschutin,  13 
Pastes,  anchovy,  67 

tomato,  67 

Pasteur,  n,  12,  14,  140 
Pasteurization,  41 

of  milk,  105 
Pellagra,  145,  215 
Penicillium,  32,  107,  143 
Peptones,  94 
Peptone  solution,  44 
Permanganate  of  potassium,  168 
Pestilence,  6 
Petri  dish,  59 

colonies,  58,  87 

culture,  57 


236 


INDEX. 


Phagocytes,  117 
Phagocytosis,  16,  117 
Phenacetin,  169 
Phenecol,  169 
Phenol,  166 
Phlogistic  zymoid,  14 
Phosphates,  soil,  94 
Phosphorescent  bacteria,  3 1 
Phragmidiothrix,  23 
Phylloxera,  186 
Physiology  of  bacteria,  28 
Pigment,  26 
Pirquet  test,  208 
Plague,  7,  212 
Plant  lice,  187 
Plasmodium,  158 
Plate  colonies,  87 

culture  counts,  67 

streak,  57 

Plates  for  counting,  88,  90 
Plating,  59 
Platinum  loop,  52 

rods,  52 

Pleomorphism,  27,  28 
Plugging  test-tubes,  37 
Pneumococcus,  135 
Pneumonia,  210 
Polar  granules,  26 
Pollender,  13 
Polymorphism,  27,  28 
Post-mortem  disinfection,  175 
Potash  zeolytes,  94 
Potato  tube  slant,  54 
Precipitins,  123 
Preservatives,  food,  181 

in  milk,  104 
Preserving  salt,  184 
Proteus,  69 
Protozoa,  156 
Prudden,  T.  M.,  20 
Pseudomonas,  23 
Psychrophile,  30 
Ptomaines,  31 
Pullman  car,  161 
Puring,  112 
Pus  formation,  117 

laudable,  13,  14,  117 
Putrid  meat,  10 
Pyre  thrum,  212 
Pyroligneous  acid,  185 

Quarantine,  177 

small-pox,  12 
Quicklime,  168 
Quinine,  158,  169 

Rabies,  15 

vaccine,  140 
Ragi,  153 
Raib,  108 
Rankin,  16 
Ratin,  in 


Rattite,  in 
Rat  virus,  in 
Ravenel,  140 
Rayner,  13 
Receptor  theory,  119 
Reagent  bottles,  36 
Receptors,  120 
Redi,  10 

Reichert  thermostat,  47 
Relapsing  fever,  156 
Resorcin,  169 
Rhizobium,  77,  95,  99 

mutabile,  61 
Rice  cooker,  45 

wine,  149 
Rindfleisch,  13 
Ring  burner,  52 
Ripening  of  cheese,  107 

of  cream,  107 
Rideal,  160 
Ringworm,  143 
Roger,  105 
Roll  culture,  55 
Root  nodules,  61,  95,  96 
Ropy  milk,  101 
Rosenau  M.  J.,  20,  105 
Rothlauf,  15 
Rotting  bacteria,  3 1 
Roux,  16 

Saccharin,  185 
Saccharomyces,  143 
Sake,.  145 

making,  149 
Salol,  169 
Salophen,  169 
Saltpeter,  31 
Salvarsan,  218 
Sanderson,  13 
Sanitation,  Mosaic,  9 
Saprophyte,  30 
Sarcina,  23 
Savage,  W.  G.,  20 
Schizomycetes,  22,  142 
Schneider,  Albert,  4 
Schroeder,  n 
Schulze,  10 
Schwann,  9 

Sedgwich-Tucker  aerobioscope,  62 
Sedimentation,  162 
Septation,  24,  28,  29 

rate  of,  34 
Sera,  for  human  use,  126 

lytic,  117 

manufacture  of,  125 

veterinary,  127 
Serum,  antitetanic,  134 

blood,  44 

humanized,  123 

Loeffler's,  43 

standardizing,  130 
Sewage  organisms,  69 
Shafer,  211 


INDEX. 


237 


Shering  lamp,  177 
Side  chain  theory,  119 
Siedentopf,  31 
Silica  hydrates,  94 
Silkworm  disease,  13 
Siloeing,  112 
Slant,  potato,  54 

tube,  51 

Sleeping  sickness,  157 
Small-pox,  ir,  211 

quarantine,  12 

vaccine,  138 
Sodium  benzoate,  184 
Soil,  bacteria  of,  59,  60 

exhaustion,  93 

fertility,  93 

formation  of,  31 

tilling  of,  94 
Soja  beans,  153 

sauce,  153 
Solution,  cleaning,  37 

normal,  49 
Solutol,  1 66 
Solveol,  1 66 
Spallanzani,  10 
Spirillaceae,  23 
Spirillae,  morphology,  25 
Spirocheta,  23,  156 
Spirochetaceae,  23 
Spirosoma,  23 
Spirillum,  23 

Spontaneous  generation,  8,  10,  11 
Spore  formation,  27 

killing  by  heat,  40 

staining,  75 
Sporulation,  24 
Spray  pump,  187 
Sprays,  187 
Squirrels,  186 
Squirrelin,  in 
Stab  culture,  56 
Staining,  70 

capsular,  76 

double,  71 

flagella,  75 

spore,  75 
Stains,  71 

acid,  70 

basic,  70 

Starter,  for  cream,  107 
Steamer,  40 
Steam  sterilizer,  39,  42 
Stegomyia,  158,  215 
Sterilization,  190 

of  culture  media,  39 

discontinuous,  41 

of  infusions,  191 
Sterilizer,  hot  air,  38 

in  pharmacy,  190 

steam,  39,  42 
Stich,  C.,  20 
Stimulins,  118 
Stitt,  E.  R.,  20] 


Streak  culture,  57 
Streptococci,  69 
Streptococcus,  23 

hollandicus,  101 

pyogenes,  141 

vaccine,  141 
Strychnin,  120 
Subcultures,  56,  87 
Sucrol,  185 
Sulphur,  26,  167,  186 

candles,  174 
Sulphuring,  9 
Sunlight,  163 

Surgical  supplies,  disinfection,  193 
Surra,  157 
Swab,  culture,  43 
"Swelling"  of  cans,  65 
Symbioses,  30,  31,  32, 
Syphilis,  157,  216 
Syrups,  64 
Szigmondy,  31 

Table,  insecticide,  188 
Tanning,  in 
Technic,  36 

Test-tube  cultures,  45,  54 
Test  tubes,  filling,  38 

plugging,  37 
Test,  Widal,  122 
Tete  fever,  157 
Tetra coccus,  23 
Theodoric,  12 
Thermal  test,  208 
ThermophUe,  30 
Thermostat,  47 
Thiothrix,  23 
Thrush,  143 
Tick  fever,  157 
Titration,  50 
Tomato  pastes,  67 
Toxalbumins,  31 
Toxemia,  34 
Toxins,  1 20 
Toxophore,  120 
Tricresol,  166 
Trichophyton,  143 
Trifolium  heterodon,  98 
Trypanosomes,  157 
Trypsin,  116 
Tsetse  fly,  157 
Tube  cultures,  87 

fermentation,  60 

slants,  51 

Tuberculins,  138,  208 
Tuberculin  test,  104 
Tuberculosis,  207 

bovine,  103 

tests,  208 

Tuberculous  cows,  103 
Tyndall,  John,  20 
Typhoid,  carriers,  101 

epidemics,  101 

fever,  209  • 


238 

Ultra  microscope,  31 
microorganisms,  31 

Vaccination,  history  of ,  n,  12 
Vaccine,  rabies,  140 

small-pox,  138 
Vaccines,  125 
Vaccinia,  12 
Van  Helmont,  8 
Varro,  12 

Vegetable  cooker,  40 
Vibrio,  69 

Vincent's  angina,  157 
Vinegar,  113 
Virus,  Danysz's,  in 

rat,  in 
Von  Dusch,  n 

Waldeyer,  13 
Walker,  160 
Warrington,  R.,  4 
Washes,  187 
Wassermann  test,  218 
Water,  copper  treatment,  179 

nitration,  181 

mineral,  64 


INDEX. 


Water,  purification  of,  179 

supplies,  179 
Webster,  Noah,  7,  20 
Weichselbaum,  15 
Widal  test,  122 
Wine,  9 

rice,  145 
Wire  baskets,  37 
Wool  bleaching,  9 
Wound  disinfection,  12 
Wright,  15 

X-rays,  163 

Yeast  cakes,  148,  154 

cells,  65 
Yeasts,  142 
Yellow  fever,  215 
Yersin's  serum,  135 
Yoghurt,  108 

tablets,  no 

Zooglea  formation,  29 
Zymoid,  phlogistic,  14 
Zymophore,  121 


// 


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D1996 

cteriology 


ltn-,5'30 


